pFind Studio: a computational solution for mass spectrometry-based proteomics

DPHL: a DIA pan-human protein mass spectrometry library for robust biomarker discovery

Genomics, proteomics & bioinformatics. 2020. Zhu, Tiansheng et al. Westlake Univ, Zhejiang Prov Lab Life Sci & Biomed, Hangzhou 310024, Peoples R China; Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou 310024, Peoples R China; Westlake Inst Adv Study, Inst Basic Med Sci, Hangzhou 310024, Peoples R China

ABSTRACT: To address the increasing need for detecting and validating protein biomarkers in clinical specimens, mass spectrometry (MS)-based targeted proteomic techniques, including the selected reaction monitoring (SRM), parallel reaction monitoring (PRM), and massively parallel dataindependent acquisition (DIA), have been developed. For optimal performance, they require the fragment ion spectra of targeted peptides as prior knowledge. In this report, we describe a MS pipeline and spectral resource to support targeted proteomics studies for human tissue samples.

To build the spectral resource, we integrated common open-source MS computational tools to assemble a freely accessible computational workflow based on Docker. We then applied the workflow to generate DPHL, a comprehensive DIA pan-human library, from 1096 data-dependent acquisition (DDA) MS raw files for 16 types of cancer samples. This extensive spectral resource was then applied to a proteomic study of 17 prostate cancer (PCa) patients. Thereafter, PRM validation was applied to a larger study of 57 PCa patients and the differential expression of three proteins in prostate tumor was validated. As a second application, the DPHL spectral resource was applied to a study consisting of plasma samples from 19 diffuse large B cell lymphoma (DLBCL) patients and 18 healthy control subjects. Differentially expressed proteins between DLBCL patients and healthy control subjects were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supports DIA and PRM MS pipelines for robust protein biomarker discovery. DPHL is freely accessible at https://www.iprox.org/page/project.html?id=IPX0001400000.

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Accelerated lysis and proteolytic digestion of biopsy-level fresh-frozen and FFPE tissue samples using pressure cycling technology

Journal of proteome research. 2020. Gao, Huanhuan et al. Westlake Inst Adv Study, Inst Basic Med Sci, Hangzhou 310024, Zhejiang, Peoples R China; Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou 310024, Zhejiang, Peoples R China

ABSTRACT: Pressure cycling technology (PCT)-assisted tissue lysis and digestion have facilitated reproducible and high-throughput proteomic studies of both fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tissue of biopsy scale for biomarker discovery. Here, we present an improved PCT method accelerating the conventional procedures by about two-fold without sacrificing peptide yield, digestion efficiency, peptide, and protein identification. The time required for processing 16 tissue samples from tissues to peptides is reduced from about 6 to about 3 h.

We analyzed peptides prepared from FFPE hepatocellular carcinoma (HCC) tissue samples by the accelerated PCT method using multiple MS acquisition methods, including short-gradient SWATH-MS, PulseDIA-MS, and 10-plex TMT-based shotgun MS. The data showed that up to 8541 protein groups could be reliably quantified from the thus prepared peptide samples. We applied the accelerated sample preparation method to 25 pairs (tumorous and matched benign) of HCC samples followed by a single-shot, 15 min gradient SWATH-MS analysis. An average of 18 453 peptides from 2822 proteins were quantified in at least 20% samples in this cohort, while 1817 proteins were quantified in at least 50% samples. The data not only identified the previously known dysregulated proteins such as MCM7, MAPRE1, and SSRP1 but also discovered promising novel protein markers, including DRAP1 and PRMT5. In summary, we present an accelerated PCT protocol that effectively doubles the throughput of PCT-assisted sample preparation of biopsy-level FF and FFPE samples without compromising protein digestion efficiency, peptide yield, and protein identification.

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Large-scale identification of N-linked intact glycopeptides in human serum using HILIC enrichment and spectral library search

Molecular & cellular proteomics : MCP. 2020. Shu, Qingbo et al. Laboratory of Protein and Peptide Pharmaceuticals & Proteomics Laboratory, Institute of Biophysics, Chinese Academy of Sciences

ABSTRACT: Large-scale identification of N-linked intact glycopeptides by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in human serum is challenging because of the wide dynamic range of serum protein abundances, the lack of a complete serum N-glycan database and the existence of proteoforms. In this regard, a spectral library search method was presented for the identification of N-linked intact glycopeptides from N-linked glycoproteins in human serum with target-decoy and motif-specific false discovery rate (FDR) control.

Serum proteins were firstly separated into low-abundance and high-abundance proteins by acetonitrile (ACN) precipitation. After digestion, the N-linked intact glycopeptides were enriched by hydrophilic interaction liquid chromatography (HILIC) and a portion of the enriched N-linked intact glycopeptides were processed by Peptide-N-Glycosidase F (PNGase F) to generate N-linked deglycopeptides. Both N-linked intact glycopeptides and deglycopeptides were analyzed by LC-MS/MS. From N-linked deglycopeptides data sets, 764 N-linked glycoproteins, 1699 N-linked glycosites and 3328 unique N-linked deglycopeptides were identified. Four types of N-linked glycosylation motifs (NXS/T/C/V, XP) were used to recognize the N-linked deglycopeptides. The spectra of these N-linked deglycopeptides were utilized for N-linked deglycopeptides library construction and identification of N-linked intact glycopeptides. A database containing 739 N-glycan masses was constructed and utilized during spectral library search for the identification of N-linked intact glycopeptides. In total, 526 N-linked glycoproteins, 1036 N-linked glycosites, 22,677 N-linked intact glycopeptides and 738 N-glycan masses were identified under 1% FDR, representing the most in-depth serum N-glycoproteome identified by LC-MS/MS at N-linked intact glycopeptide level. Copyright © 2020 © 2020 Shu et al. Published by Elsevier Inc. All rights reserved.

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Global chemical modifications comparison of human plasma proteome from two different age groups

Scientific Reports. 2020. Liu, Yongtao et al. Beijing Normal Univ, Dept Biochem & Mol Biol, Beijing Key Lab Gene Engn Drug & Biotechnol, Beijing, Peoples R China

ABSTRACT: In this study, two groups of human plasma proteome at different age groups (old and young) were used to perform a comparison of global chemical modifications, as determined by tandem mass spectrometry (MS/MS) combined with non-limiting modification identification algorithms. The sulfhydryl in the cysteine A total of 4 molecular modifications were found to have significant differences passing random grouping tests: the succinylation and phosphorylation modification of cysteine (Cys, C) and the modification of lysine (Lys, K) with threonine (Thr, T) were significantly higher in the old group than in the young group, while the carbamylation of lysine was lower in the young group.

We speculate that there is an increase in certain modified proteins in the blood of the old people which, in turn, changes the function of those proteins. This change may be one of the reasons why old people are more likely than young people to be at risk for age-related diseases, such as metabolic diseases, cerebral and cardiovascular diseases, and cancer.

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An innovative artificial photosystem II constructed from PSII core of Thermosynechococcus vulcanus and LHCII of Pisum sativum-A new approach for studying the function of photosynthetic antenna

Plant Physiology and Biochemistry. 2020. Li, Ling et al. Chinese Acad Sci, Inst Bot, Key Lab Plant Resources, Beijing Bot Garden, Beijing 100093, Peoples R China; Chinese Acad Sci, Qingdao Inst, Shanghai Inst Technol Phys, Binhai Rd 50, Qingdao 264000, Peoples R China

ABSTRACT: In photosynthesis, the antenna system captures solar energy and transfers the excitations to photosystem II (PSII) core complex where charge separation, water splitting and oxygen evolution occur. In the evolution of photosynthesis from aquatic to terrestrial environments, the structure of PSII core complex was highly conserved while a variety of antenna forms became differentiated. In order to study the principles for energy transport from antenna to the PSII reaction center, we have explored whether the major light harvesting complex of PSII (LHCII) of higher plants can transfer energy to the cyanobacteria PSII core complexes (CC).

For this purpose, LHCII from pea and CC from Thermosynechococcus vulcanus were isolated and co-reconstituted into liposome at LHCII:CC molar ratios of 2:1, 4:1 and 6:1, respectively. Chemical-cross linking followed by LC-MS/MS analysis confirmed the biochemical interaction between LHCII and CC in the liposome membrane. The analyses of 77 K fluorescence emission spectra and antenna cross section of PSII indicated that LHCII can transfer energy directly to the cyanobacterial CC. The study has laid the basis for further research on the mechanism of energy transfer from LHCII to PSII CC. This result may also open a new possibility for design and development of new artificial PSII in the application of solar energy conversion.

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健康人的尿液中为什么会有蛋白质?

中国科学. 生命科学. 2020. 刘永涛 et al. 北京医院国家老年医学中心检验医学科, 北京 100730, 中国; 北京师范大学生命科学学院, 生物化学与分子生物学系, 基因工程药物及生物技术北京市重点实验室, 北京 100875, 中国

ABSTRACT: Proteins are essential nutrients for humans, but why is there a small amount of proteins excreted through urine? Although healthy people have low levels of protein in the urine, they are rich in variety.The proteins in them may be unnecessary wastes of the body or may even have a toxic effect on the body and must be excreted.Compared with small-molecule metabolites, proteins have a complex structure and various functions.Even a small change at the molecular level will affect their subsequent biological functions.A comprehensive comparison of the molecular level modifications of plasma and urine proteome, the difference may provide clues to the presence of protein in the urine.In this study, a total of 9 healthy people's urine and 9 healthy people's plasma samples were collected.The samples were analyzed by high-resolution tandem mass spectrometry and unlabeled quantitative proteomics techniques, and the non-limiting modification identification algorithm was used to to compare molecular modifications between two types of samples as a whole.The results showed that the amount of cysteine(Cys)modification to dehydroalanine(Dha)in the urine proteome was higher than that in plasma.The molecular modification[CysDha] destroys the disulfide bond of the original protein,thereby affecting or even changing the structure and biological function of the protein.Therefore, this study revealed the differences in the overall proteome modification of plasma and urine and pointed out that the structure of protein may be irreversibly changed due to the difference in modification of protein molecules, which in turn caused proteins which lost their functions and even toxic proteins from the blood to be excreted into the urine.

Persulfidation-based modification of cysteine desulfhydrase and the NADPH oxidase RBOHD controls guard cell abscisic acid signaling

The Plant Cell. 2020. Shen, J et al. Nanjing Agr Univ, Coll Life Sci, Lab Ctr Life Sci, Nanjing 210095, Peoples R China.

ABSTRACT: A persulfidation-based reversible post-translational modification of Cys desulfhydrase and NADPH oxidase RBOHD fine-tunes guard cell ABA signaling. Hydrogen sulfide (H2S) is a gaseous signaling molecule that regulates diverse cellular signaling pathways through persulfidation, which involves the post-translational modification of specific Cys residues to form persulfides. However, the mechanisms that underlie this important redox-based modification remain poorly understood in higher plants. We have, therefore, analyzed how protein persulfidation acts as a specific and reversible signaling mechanism during the abscisic acid (ABA) response in Arabidopsis (Arabidopsis thaliana).

Here we show that ABA stimulates the persulfidation of l-CYSTEINE DESULFHYDRASE1, an important endogenous H2S enzyme, at Cys44 and Cys205 in a redox-dependent manner. Moreover, sustainable H2S accumulation drives persulfidation of the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN D (RBOHD) at Cys825 and Cys890, enhancing its ability to produce reactive oxygen species. Physiologically, s-persulfidation-induced RBOHD activity is relevant to ABA-induced stomatal closure. Together, these processes form a negative feedback loop that fine-tunes guard cell redox homeostasis and ABA signaling. These findings not only expand our current knowledge of H2S function in the context of guard cell ABA signaling, but also demonstrate the presence of a rapid signal integration mechanism involving specific and reversible redox-based post-translational modifications that occur in response to changing environmental conditions.

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Accurate annotation of human protein-coding small open reading frames

Nature chemical biology. 2020. Martinez, TF et al. Salk Inst Biol Studies, Clayton Fdn Labs Peptide Biol, 10010 N Torrey Pines Rd, La Jolla, CA 92037 USA.

ABSTRACT: Functional protein-coding small open reading frames (smORFs) are emerging as an important class of genes. However, the number of translated smORFs in the human genome is unclear because proteogenomic methods are not sensitive enough, and, as we show, Ribo-seq strategies require additional measures to ensure comprehensive and accurate smORF annotation. Here, we integrate de novo transcriptome assembly and Ribo-seq into an improved workflow that overcomes obstacles with previous methods, to more confidently annotate thousands of smORFs.

Evolutionary conservation analyses suggest that hundreds of smORF-encoded microproteins are likely functional. Additionally, many smORFs are regulated during fundamental biological processes, such as cell stress. Peptides derived from smORFs are also detectable on human leukocyte antigen complexes, revealing smORFs as a source of antigens. Thus, by including additional validation into our smORF annotation workflow, we accurately identify thousands of unannotated translated smORFs that will provide a rich pool of unexplored, functional human genes. An improved workflow combining de novo transcriptome assembly and Ribo-seq validated by cellular antigen display is developed to maximize small peptide discovery, leading to identification of thousands of unannotated protein-coding smORFs.

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A streamlined mass spectrometry--based proteomics workflow for large-scale FFPE tissue analysis

The Journal of pathology. 2020. Coscia, F et al. Max Planck Inst Biochem, D-82152 Martinsried, Germany.

ABSTRACT: Formalin fixation and paraffin-embedding (FFPE) is the most common method to preserve human tissue for clinical diagnosis, and FFPE archives represent an invaluable resource for biomedical research. Proteins in FFPE material are stable over decades but their efficient extraction and streamlined analysis by mass spectrometry (MS)-based proteomics has so far proven challenging. Herein we describe a MS-based proteomic workflow for quantitative profiling of large FFPE tissue cohorts directly from histopathology glass slides.

We demonstrate broad applicability of the workflow to clinical pathology specimens and variable sample amounts, including low-input cancer tissue isolated by laser microdissection. Using state-of-the-art data dependent acquisition (DDA) and data independent acquisition (DIA) MS workflows, we consistently quantify a large part of the proteome in 100 min single-run analyses. In an adenoma cohort comprising more than 100 samples, total workup took less than a day. We observed a moderate trend towards lower protein identification in long-term stored samples (>15 years), but clustering into distinct proteomic subtypes was independent of archival time. Our results underscore the great promise of FFPE tissues for patient phenotyping using unbiased proteomics and they prove the feasibility of analyzing large tissue cohorts in a robust, timely, and streamlined manner. (c) 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

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Pervasive translation of circular RNAs driven by short IRES-like elements

Nature communications. 2020. Fan, XJ et al. Shanghai Inst Nutr & Hlth, Biomed Big Data Ctr, CAS Ctr Excellence Mol Cell Sci, CAS Key Lab Computat Biol, Shanghai, Peoples R China; Chinese Acad Sci, Univ Chinese Acad Sci, Beijing, Peoples R China

ABSTRACT: Some circular RNAs (circRNAs) were found to be translated through IRES-driven mechanism, however the scope and functions of circRNA translation are unclear because endogenous IRESs are rare. To determine the prevalence and mechanism of circRNA translation, we develop a cell-based system to screen random sequences and identify 97 overrepresented hexamers that drive cap-independent circRNA translation. These IRES-like short elements are significantly enriched in endogenous circRNAs and sufficient to drive circRNA translation.

We further identify multiple trans-acting factors that bind these IRES-like elements to initiate translation. Using mass-spectrometry data, hundreds of circRNA-coded peptides are identified, most of which have low abundance due to rapid degradation. As judged by mass-spectrometry, 50% of translatable endogenous circRNAs undergo rolling circle translation, several of which are experimentally validated. Consistently, mutations of the IRES-like element in one circRNA reduce its translation. Collectively, our findings suggest a pervasive translation of circRNAs, providing profound implications in translation control.Unbiased screen of random sequences identified many short IRES-like elements to drive circular RNA translation and hundreds of rolling circle translation events, suggesting a pervasive cap-independent translation in human transcriptome.

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Cancer neoantigen prioritization through sensitive and reliable proteogenomics analysis

Nature communications. 2020. Wen, B et al. Baylor Coll Med, Lester & Sue Smith Breast Ctr, Houston, TX 77030 USA.

ABSTRACT: Genomics-based neoantigen discovery can be enhanced by proteomic evidence, but there remains a lack of consensus on the performance of different quality control methods for variant peptide identification in proteogenomics. We propose to use the difference between accurately predicted and observed retention times for each peptide as a metric to evaluate different quality control methods. To this end, we develop AutoRT, a deep learning algorithm with high accuracy in retention time prediction. Analysis of three cancer data sets with a total of 287 tumor samples using different quality control strategies results in substantially different numbers of identified variant peptides and putative neoantigens.

Our systematic evaluation, using the proposed retention time metric, provides insights and practical guidance on the selection of quality control strategies. We implement the recommended strategy in a computational workflow named NeoFlow to support proteogenomics-based neoantigen prioritization, enabling more sensitive discovery of putative neoantigens. Identifying mutation-derived neoantigens by proteogenomics requires robust strategies for quality control. Here, the authors propose peptide retention time as an evaluation metric for proteogenomics quality control methods, and develop a deep learning algorithm for accurate retention time prediction.

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Structural snapshots of human pre-60S ribosomal particles before and after nuclear export

Nature communications. 2020. Liang, XM et al. Peking Univ, Peking Tsinghua Joint Ctr Life Sci, Sch Life Sci, State Key Lab Membrane Biol, Beijing 100871, Peoples R China.

ABSTRACT: Ribosome biogenesis is an elaborate and energetically expensive program that involve two hundred protein factors in eukaryotes. Nuclear export of pre-ribosomal particles is one central step which also serves as an internal structural checkpoint to ensure the proper completion of nuclear assembly events. Here we present four structures of human pre-60S particles isolated through a nuclear export factor NMD3, representing assembly stages immediately before and after nuclear export. These structures reveal locations of a dozen of human factors, including an uncharacterized factor TMA16 localized between the 5S RNA and the P0 stalk.

Comparison of these structures shows a progressive maturation for the functional regions, such as peptidyl transferase centre and peptide exit tunnel, and illustrate a sequence of factor-assisted rRNA maturation events. These data facilitate our understanding of the global conservation of ribosome assembly in eukaryotes and species-specific features of human assembly factors. Ribosome biogenesis in eukaryotes is a complex process that involves more than 200 protein factors. Here the authors present a structural analysis of a collection of human pre-60S structures sampled through a nuclear export adaptor NMD3, representing structural snapshots of pre-60S particles immediately before and after passing through nuclear pore complex.

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Improvements on the quantitative analysis of Trypanosoma cruzi histone post translational modifications: Study of changes in epigenetic marks through the parasite's metacyclogenesis and life cycle

Journal of proteomics. 2020. de Lima, LP et al. Inst Butantan, Lab Especial Ciclo Celular, Ave Vital Brasil 1500, BR-05503900 Sao Paulo, Brazil.

ABSTRACT: Trypanosome histone N-terminal sequences are very divergent from the other eukaryotes, although they are still decorated by post-translational modifications (PTMs). Here, we used a highly robust workflow to analyze histone PTMs in the parasite Trypanosoma cruzi using mass spectrometry-based (MS-based) data-independent acquisition (DIA). We adapted the workflow for the analysis of the parasite's histone sequences by modifying the software EpiProfile 2.0, improving peptide and PTM quantification accuracy.

This workflow could now be applied to the study of 141 T. cruzi modified histone peptides, which we used to investigate the dynamics of histone PTMs along the metacyclogenesis and the life cycle of T. cruzi. Global levels of histone acetylation and methylation fluctuates along metacyclogenesis, however most critical differences were observed between parasite life forms. More than 66 histone PTM changes were detected. Strikingly, the histone PTM pattern of metacyclic trypomastigotes is more similar to epimastigotes than to cellular trypomastigotes. Finally, we highlighted changes at the H4 N-terminus and at H3K76 discussing their impact on the trypanosome biology. Altogether, we have optimized a workflow easily applicable to the analysis of histone PTMs in T. cruzi and generated a dataset that may shed lights on the role of chromatin modifications in this parasite. Significance: Trypanosomes are unicellular parasites that have divergent histone sequences, no chromosome condensation and a peculiar genome/gene regulation. Genes are transcribed from divergent polycistronic regions and post-transcriptional gene regulation play major role on the establishment of transcripts and protein levels. In this regard, the fact that their histones are decorated with multiple PTMs raises interesting questions about their role. Besides, this digenetic organism must adapt to different environments changing its metabolism accordingly. As metabolism and epigenetics are closely related, the study of histone PTMs in trypanosomes may enlighten this strikingly, and not yet fully understood, interplay. From a biomedical perspective, the comprehensive study of molecular mechanisms associated to the metacyclogenesis process is essential to create better strategies for controlling Chagas disease.

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Atg1 kinase in fission yeast is activated by Atg11-mediated dimerization and cis-autophosphorylation

Elife. 2020. Pan, ZQ et al. Natl Inst Biol Sci, Beijing, Peoples R China.

ABSTRACT: Autophagy is a proteolytic pathway that is conserved from yeasts to mammals. Atg1 kinase is essential for autophagy, but how its activity is controlled remains insufficiently understood. Here, we show that, in the fission yeast Schizosaccharomyces pombe, Atg1 kinase activity requires Atg11, the ortholog of mammalian FIP200/RB1CC1, but does not require Atg13, Atg17, or Atg101. Remarkably, a 62 amino acid region of Atg11 is sufficient for the autophagy function of Atg11 and for supporting the Atg1 kinase activity.

This region harbors an Atg1-binding domain and a homodimerization domain. Dimerizing Atg1 is the main role of Atg11, as it can be bypassed by artificially dimerizing Atg1. In an Atg1 dimer, only one Atg1 molecule needs to be catalytically active, suggesting that Atg1 activation can be achieved through cis-autophosphorylation. We propose that mediating Atg1 oligomerization and activation may be a conserved function of Atg11/FIP200 family proteins and cis-autophosphorylation may be a general mechanism of Atg1 activation.

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Global lysine crotonylation profiling of mouse liver

Proteomics. 2020. Liu, JF et al. Chinese Acad Med Sci & Peking Union Med Coll, Inst Basic Med Sci, Dept Biochem & Mol Biol, State Key Lab Med Mol Biol, Beijing 100005, Peoples R China.

ABSTRACT: Lysine crotonylation (Kcr) is a recently discovered post-translational modification that potentially regulates multiple biological processes. With an objective to expand the available crotonylation datasets, LC-MS/MS is performed using mouse liver samples under normal physiological conditions to obtain in vivo crotonylome. A label-free strategy is used and 10 034 Class I (localization probabilities > 0.75) crotonylated sites are identified in 2245 proteins. The KcrE, KcrD, and EKcr motifs are significantly enriched in the crotonylated peptides.

The identified crotonylated proteins are mostly enzymes and primarily located in the cytoplasm and nucleus. Functional enrichment analysis based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes shows that the crotonylated proteins are closely related to the purine-containing compound metabolic process, ribose phosphate metabolic process, carbon metabolism pathway, ribosome pathway, and a series of metabolism-associated biological processes. To the best of the authors' knowledge, this research provides the first report on the mouse liver crotonylome. Furthermore, it offers additional evidence that crotonylation exists in non-histone proteins, and is likely involved in various biological processes. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium with the dataset identifiers PXD019145.

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Orally efficacious broad-spectrum allosteric inhibitor of paramyxovirus polymerase

Nature microbiology. 2020. Cox, RM et al. Georgia State Univ, Inst Biomed Sci, Atlanta, GA 30303 USA.

ABSTRACT: Paramyxoviruses such as human parainfluenza virus type-3 (HPIV3) and measles virus (MeV) are a substantial health threat. In a high-throughput screen for inhibitors of HPIV3 (a major cause of acute respiratory infection), we identified GHP-88309-a non-nucleoside inhibitor of viral polymerase activity that possesses unusual broad-spectrum activity against diverse paramyxoviruses including respiroviruses (that is, HPIV1 and HPIV3) and morbilliviruses (that is, MeV). Resistance profiles of distinct target viruses overlapped spatially, revealing a conserved binding site in the central cavity of the viral polymerase (L) protein that was validated by photoaffinity labelling-based target mapping.

Mechanistic characterization through viral RNA profiling and in vitro MeV polymerase assays identified a block in the initiation phase of the viral polymerase. GHP-88309 showed nanomolar potency against HPIV3 isolates in well-differentiated human airway organoid cultures, was well tolerated (selectivity index > 7,111) and orally bioavailable, and provided complete protection against lethal infection in a Sendai virus mouse surrogate model of human HPIV3 disease when administered therapeutically 48 h after infection. Recoverees had acquired robust immunoprotection against reinfection, and viral resistance coincided with severe attenuation. This study provides proof of the feasibility of a well-behaved broad-spectrum allosteric antiviral and describes a chemotype with high therapeutic potential that addresses major obstacles of anti-paramyxovirus drug development. In this Article, the authors report the discovery of a small molecule (GHP-88309) that is a new class of allosteric viral polymerase inhibitor that works against two different genera of paramyxoviruses.

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A protein identification algorithm optimization for mass spectrometry data using deep learning

2020 3rd International Conference on Advanced Electronic Materials, Computers and Software Engineering (AEMCSE). 2020. Xu, Rui et al. State Key Laboratory of Proteomics, Beijing Proteome Research Center National Center for Protein Sciences (Beijing), Beijing

ABSTRACT: Protein sequence database search is one of the most commonly used methods for protein identification in shotgun proteomics. In tradition, searching a protein sequence database is usually required to construct the theoretical spectrum for each peptide at first, which only considers the information of mass-to-charge ratio at present. However, the information related to isotope peak intensity is neglected. Thanks to the rapid development of artificial intelligence technique in recent years, deep learning-based MS/MS spectrum prediction tools have showed a high accuracy and great potentials to improve the sensitivity and accuracy of protein sequence database searching.

In this study, we used a deep learning model (pDeep2) to predict the theoretical mass spectrum of all peptides and applied it to a database searching tool (DeepNovo), thus improving the sensitivity and accuracy of peptide identification.

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An Algorithm to Improve the Speed of Semi and Non-specific Enzyme Searches in Proteomics

Current bioinformatics. 2020. Rolfs, Z et al. Univ Wisconsin, Dept Chem, 1101 Univ Ave, Madison, WI 53706 USA.

ABSTRACT: Background: The identification of non-specifically cleaved peptides in proteomics and peptidomics poses a significant computational challenge. Current strategies for the identification of such peptides are typically time-consuming and hinder routine data analysis. Objective: We aimed to design an algorithm that would improve the speed of semi- and nonspecific enzyme searches and could be applied to existing search programs. Methods: We developed a novel search algorithm that leverages fragment-ion redundancy to simultaneously search multiple non-specifically cleaved peptides at once.

Briefly, a theoretical peptide tandem mass spectrum is generated using only the fragment-ion series from a single terminus. This spectrum serves as a proxy for several shorter theoretical peptides sharing the same terminus. After database searching, amino acids are removed from the opposing terminus until the observed and theoretical precursor masses match within a given mass tolerance. Results: The algorithm was implemented in the search program MetaMorpheus and found to perform an order of magnitude faster than the traditional MetaMorpheus search and produce superior results. Conclusion: We report a speedy non-specific enzyme search algorithm that is open-source and enables search programs to utilize fragmention redundancy to achieve a notable increase in search speed.

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Characterization of Lysine Monomethylome and Methyltransferase in Model Cyanobacterium Synechocystis sp. PCC 6803

Genomics, proteomics & bioinformatics. 2020. Lin, XH et al. Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China.

ABSTRACT: Protein lysine methylation is a prevalent post-translational modification (PTM) and plays critical roles in all domains of life. However, its extent and function in photosynthetic organisms are still largely unknown. Cyanobacteria are a large group of prokaryotes that carry out oxygenic photosynthesis and are applied extensively in studies of photosynthetic mechanisms and environmental adaptation. Here we integrated propionylation of monomethylated proteins, enrichment of the modified peptides, and mass spectrometry (MS) analysis to identify monomethylated proteins in Synechocystis sp.

PCC 6803 (Synechocystis). Overall, we identified 376 monomethylation sites in 270 proteins, with numerous monomethylated proteins participating in photosynthesis and carbon metabolism. We subsequently demonstrated that CpcM, a previously identified asparagine methyl-transferase in Synechocystis, could catalyze lysine monomethylation of the potential aspartate amino-transferase 5110480 both in vivo and in vitro and regulate the enzyme activity of 5110480. The loss of CpcM led to decreases in the maximum quantum yield in primary photosystem II (PSII) and the efficiency of energy transfer during the photosynthetic reaction in Synechocystis. We report the first lysine monomethylome in a photosynthetic organism and present a critical database for functional analyses of monomethylation in cyanobacteria. The large number of monomethylated proteins and the identification of CpcM as the lysine methyltransferase in cyanobacteria suggest that reversible methylation may influence the metabolic process and photosynthesis in both cyanobacteria and plants.

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SlFERL interacts with S-adenosylmethionine synthetase to regulate fruit ripening

Plant Physiology. 2020. Ji, DC et al. Chinese Acad Sci, Innovat Acad Seed Design, Inst Bot, Key Lab Plant Resources, Beijing 100093, Peoples R China.

ABSTRACT: The tomato membrane protein SlFERL regulates fruit ripening via modulating ethylene production. Fruit ripening is a complex and genetically programmed process modulated by transcription factors, hormones, and other regulators. However, the mechanism underlying the regulatory loop involving the membrane-protein targets of RIPENING-INHIBITOR (RIN) remains poorly understood. To unravel the function of tomato (Solanum lycopersicum) FERONIA Like (SlFERL), a putative MADS-box transcription factor target gene, we investigated and addressed the significance of SlFERL in fruit ripening by combining reverse genetics, biochemical, and cytological analyses.

Here, we report that RIN and Tomato AGAMOUS-LIKE1 (TAGL1) directly bind to the promoter region of SlFERL and further activate its expression transcriptionally, suggesting a potential role of SlFERL in fruit ripening. Overexpression of SlFERL significantly accelerated the ripening process of tomato fruit, whereas RNA interference knockdown of SlFERL resulted in delayed fruit ripening. Moreover, a surface plasmon resonance assay coupled with tandem mass spectrometry and a protein interaction assay revealed that SlFERL interacts with the key enzyme S-adenosyl-Met synthetase 1 (SlSAMS1) in the ethylene biosynthesis pathway, leading to increased S-adenosyl-Met accumulation and elevated ethylene production. Thus, SlFERL serves as a positive regulator of ethylene production and fruit ripening. This study provides clues to the molecular regulatory networks underlying fruit ripening.

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Unambiguous Phosphosite Localization through the Combination of Trypsin and LysargiNase Mirror Spectra in a Large-Scale Phosphoproteome Study

Journal of proteome research. 2020. Xu, F et al. Beijing Inst Life, Natl Ctr Prot Sci Beijing, Beijing Proteome Res Ctr, State Key Lab Prote, Beijing 102206, Peoples R China.

ABSTRACT: Understanding of the kinase-guided signaling pathways requires the identification and analysis of phosphosites. Mass spectrometry (MS)-based phosphoproteomics is a rapid and highly sensitive approach for high-throughput identification of phosphosites. However, phosphosite determination from MS data with a single protease is more likely to be ambiguous, regardless of the strategy used for phosphopeptide detection. Here, we explored the application of LysargiNase, which was recently reported to mirror trypsin in specificity to cleave arginine and lysine residues exclusively at the N-terminal side.

We found that the combination of trypsin and LysargiNase mirror spectra resulted in higher ion coverage in MS(2 )spectra. The median ion coverage values of b ions in tryptic spectra, LysargiNase spectra, and combined spectra are 8.3, 20.5, and 25.0%, respectively. As for the median ion coverage of y ions, these values are 27.8, 10.0, and 32.3%. Higher ion coverage was helpful to pinpoint the precise phosphosites. Compared to trypsin alone, the combined use of trypsin and LysargiNase mirror spectra enabled 67.1% of mirror spectra with unreliable scores (confidence score <0.75) to become reliable (confidence score >= 0.75). Meanwhile, all of the mirror peptide-spectrum matches (PSMs) with multiple potential phosphosites from trypsin and LysargiNase digests could be assigned one precise phosphosite after applying the combination strategy. Besides, the combination strategy could identify more novel phosphosites than the union strategy did. We synthesized three phosphopeptides corresponding to the three novel phosphosites and validated the reliability of the identification. Taken together, our data demonstrated the distinctive potential of the combination strategy presented here for unambiguous phosphosite localization (Project accession PXD011178).

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Global characterization of modifications to the charge isomers of IgG antibody

Journal of Pharmaceutical Analysis. 2020. Cui, XL et al. State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing

ABSTRACT: Posttranslational modifications of antibody products affect their stability, charge distribution, and drug activity and are thus a critical quality attribute. The comprehensive mapping of antibody modifications and different charge isomers (CIs) is of utmost importance but is challenging. We intended to quantitatively characterize the posttranslational modification status of CIs of antibody drugs and explore the impact of posttranslational modifications on charge heterogeneity. The CIs of antibodies were fractionated by strong cation exchange chromatography and verified by capillary isoelectric focusing-whole column imaging detection, followed by stepwise structural characterization at three levels.

First, the differences between CIs were explored at the intact protein level using a top-down mass spectrometry approach; this showed differences in glycoforms and deamidation status. Second, at the peptide level, common modifications of oxidation, deamidation, and glycosylation were identified. Peptide mapping showed nonuniform deamidation and glycoform distribution among CIs. In total, 10 N-glycoforms were detected by peptide mapping. Finally, an in-depth analysis of glycan variants of CIs was performed through the detection of enriched glycopeptides. Qualitative and quantitative analyses demonstrated the dynamics of 24 N-glycoforms. The results revealed that sialic acid modification is a critical factor accounting for charge heterogeneity, which is otherwise missed in peptide mapping and intact molecular weight analyses. This study demonstrated the importance of the comprehensive analyses of antibody CIs and provides a reference method for the quality control of biopharmaceutical analysis.

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UPEFinder: A Bioinformatic Tool for the Study of Uncharacterized Proteins Based on Gene Expression Correlation and the PageRank Algorithm

Journal of proteome research. 2020. Gonzalez-Gomariz, J et al. Navarra Inst Hlth Res, IdiSNA, E-31008 Pamplona, Spain.

ABSTRACT: The Human Proteome Project (HPP) is leading the international effort to characterize the human proteome. Although the main goal of this project was first focused on the detection of missing proteins, a new challenge arose from the need to assign biological functions to the uncharacterized human proteins and describe their implications in human diseases. Not only the proteins with experimental evidence (uPE1 proteins) but also the uncharacterized missing proteins (uMPs) were the objects of study in this challenge, neXt-CP50.

In this work, we developed a new bioinformatic approach to infer biological annotations for the uPE1 proteins and uMPs based on a "guilt-by-association" analysis using public RNA-Seq data sets. We used the correlation of these proteins with the well-characterized PE1 proteins to construct a network. In this way, we applied the PageRank algorithm to this network to identify the most relevant nodes, which were the biological annotations of the uncharacterized proteins. All of the generated information was stored in a database. In addition, we implemented the web application UPEFinder (https://upefinder. proteored.org ) to facilitate the access to this new resource. This information is especially relevant for the researchers of the HPP who are interested in the generation and validation of new hypotheses about the functions of these proteins. Both the database and the web application are publicly available (https://github.com/tibioinformat/UPEfinder).

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Quantitative Perspective on Online Flow Reaction Profiling Using a Miniature Mass Spectrometer

ORGANIC PROCESS RESEARCH & DEVELOPMENT. 2020. Sheng, HM et al. Merck & Co Inc, Analyt Sci, Rahway, NJ 07065 USA.

ABSTRACT: Online mass spectrometry has proven to be a useful tool for characterizing many aspects of chemical reactions. However, to the best of the authors' knowledge, no reference standard (RS) quantitation approach has been applied in online MS profiling work to date. In this study, we present a RS approach for online quantitation of an aerobic oxidation reaction in flow using a miniature mass spectrometer, with both internal RS and external RS quantitation approaches being evaluated. Quinoline, a structurally similar and chemically inert compound under these reaction conditions, was chosen as the RS to quantify the pyridine aldehyde product.

To investigate the optimal RS concentration and instrument attenuation, calibration curves were established by plotting the product/RS peak intensity ratio against the theoretical product yield at different attenuation (dilution factor) values. The MS quantitation results for the actual flow reactions were validated with conventional offline H-1 NMR analysis.

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Open-pFind Verified Four Missing Proteins from Multi-Tissues

Journal of proteome research. 2020. Wu, SJ et al. Wuhan Univ, Sch Basic Med Sci, Minist Educ, Key Lab Combinatl Biosynth & Drug Discovery, Wuhan 430072, Peoples R China.

ABSTRACT: The Chromosome-Centric Human Proteome Project (C-HPP) was launched in 2012 to perfect the annotation of human protein existence by identifying stronger evidence of the expression of missing proteins (MPs) at the protein level. After an 8 year effort all over the world, the number of MPs in the neXtProt database significantly decreased from 5511 (2012-02-24) to 1899 (2020-01-17). It is now more difficult to provide confident evidence of the remaining MPs because of their specific characteristics, including low abundance, low molecular weight, unexpected modifications, transmembrane structure, tissue-expression specificity, and so on.

A higher resolution mass spectrometry (MS) interpretation engine might provide an opportunity to identify these buried MPs in complex samples by the combination with multi-tissue large-scale proteomics. In this study, open-pFind was used to dig MPs from 20 pairs of healthy human tissues by Wang et al. ( Mol. Syst. Biol. 2019, 15 (2), e8503) combined with our large-scale testis data set digested by three enzymes (Glu-C, Lys-C, and trypsin) with specificity for different amino acid residues ( J. Proteme Res. 2019, 18 (12), 4189-4196). A total of 1 535 536 peptides with 17 283 477 peptide-spectrum matches (PSMs) were mapped to 14 279 protein entries at a false discovery rate of <1% at the PSM, peptide, and protein levels. A total of 103 MP candidates were identified, among which 86 candidates had more unique peptide numbers compared with our single testis tissue. After rigorous screening, manual checks, peptide synthesis, and matching with documented peptides from PeptideAtlas, we validated four MPs, P0C7T8 (duodenum and small intestine), Q8WWZ4 (stomach and rectum), Q8IV35 (fallopian tube), and O14921 (tonsil), at the protein level. All MS raw files have been deposited to the ProteomeXchange with identifier PXD021391.

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D283 Med, a Cell Line Derived from Peritoneal Metastatic Medulloblastoma: A Good Choice for Missing Protein Discovery

Journal of proteome research. 2020. Zhang, YL et al. BGI Shenzhen, BGI Genom, Shenzhen 518083, Guangdong, Peoples R China.

ABSTRACT: Since the Chromosome-Centric Human Proteome Project (C-HPP) was launched in 2010, many techniques have been adopted for the discovery of missing proteins (MPs). Because of these efforts, only 1481 MPs remained as of July 2020; however, by relying only on technique optimization, researchers have reached a bottleneck in MP discovery. Protein expression is tissue- or cell-type-dependent. The tissues of the human testis and brain have been reported to harbor a large number of tissue-specific genes and proteins; however, few studies have been performed on human brain tissue or cells to identify MPs.

Herein a metastatic cell line derived from brain cancer, D283 Med, was used to search for MPs. With a traditional and simple shotgun workflow to separate the peptides into 20 fractions, 12 MPs containing at least two unique non-nested peptides (amino acid length >= 9) were identified in this cell line with a protein false discovery rate of <1%. Following the same experimental protocol, only one MP was found in a nonmetastatic brain cancer cell line, U-118 MG. Furthermore, 12 MPs were verified as having two non-nested unique peptides by matching them with corresponding chemically synthesized peptides through parallel reaction monitoring. These results clearly demonstrate that the appropriate selection of experimental materials, either tissues or cell lines, is imperative for MP discovery. The data obtained in this study are available via ProteomeXchange (PXD021482) and PeptideAtlas (PASS01627).

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Open Search Strategy for Inferring the Masses of Cross-Link Adducts on Proteins

Analytical Chemistry. 2020. Slavin, M et al. Hebrew Univ Jerusalem, Inst Life Sci, IL-9190401 Jerusalem, Israel.

ABSTRACT: Development of new reagents for protein cross-linking is constantly ongoing. The chemical formulas for the linker adducts formed by these reagents are usually deduced from expert knowledge and then validated by mass spectrometry. Clearly, it would be more rigorous to infer the chemical compositions of the adducts directly from the data without any prior assumptions on their chemistries. Unfortunately, the analysis tools that are currently available to detect chemical modifications on linear peptides are not applicable to the case of two cross-linked peptides.

Here, we show that an adaptation of the open search strategy that works on linear peptides can be used to characterize cross-link modifications in pairs of peptides. We benchmark our approach by correctly inferring the linker masses of two well-known reagents, DSS and formaldehyde, to accuracies of a few parts per million. We then investigate the cross-linking chemistries of two poorly characterized reagents: EMCS and glutaraldehyde. In the case of EMCS, we find that the expected cross-linking chemistry is accompanied by a competing chemistry that targets other amino acid types. In the case of glutaraldehyde, we find that the chemical formula of the dominant linker is C5H4, which indicates a ringed aromatic structure. These results demonstrate how, with very little effort, our approach can yield nontrivial insights to better characterize new cross-linkers.

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Pre-termination transcription complex: structure and function

Molecular cell. 2020. Hao, ZT et al. NYU, Sch Med, Dept Biochem & Mol Pharmacol, New York, NY 10016 USA.

ABSTRACT: Rho is a general transcription termination factor playing essential roles in RNA polymerase (RNAP) recycling, gene regulation, and genomic stability in most bacteria. Traditional models of transcription termination postulate that hexameric Rho loads onto RNA prior to contacting RNAP and then translocates along the transcript in pursuit of the moving RNAP to pull RNA from it. Here, we report the cryoelectron microscopy (cryo-EM) structures of two termination process intermediates. Prior to interacting with RNA, Rho forms a specific "pre-termination complex'' (PTC) with RNAP and elongation factors NusA and NusG, which stabilize the PTC.

RNA exiting RNAP interacts with NusA before entering the central channel of Rho from the distal C-terminal side of the ring. We map the principal interactions in the PTC and demonstrate their critical role in termination. Our results support a mechanism in which the formation of a persistent PTC is a prerequisite for termination.

[more...]

Synergistic optimization of Liquid Chromatography and Mass Spectrometry parameters on Orbitrap Tribrid mass spectrometer for high efficient data-dependent proteomics

Journal of Mass Spectrometry. 2020. Huang, PW et al. Southern Univ Sci & Technol, Dept Chem, Shenzhen 518055, Peoples R China.

ABSTRACT: Steady improvement in Orbitrap-based mass spectrometry (MS) technologies has greatly advanced the peptide sequencing speed and depth. In-depth analysis of the performance of state-of-the-art MS and optimization of key parameters can improve sequencing efficiency. In this study, we first systematically compared the performance of two popular data-dependent acquisition approaches, with Orbitrap as the first-stage (MS1) mass analyzer and the same Orbitrap (high-high approach) or ion trap (high-low approach) as the second-stage (MS2) mass analyzer, on the Orbitrap Fusion mass spectrometer.

High-high approach outperformed high-low approach in terms of better saturation of the scan cycle and higher MS2 identification rate. However, regardless of the acquisition method, there are still more than 60% of peptide features untargeted for MS2 scan. We then systematically optimized the MS parameters using the high-high approach. Increasing the isolation window in the high-high approach could facilitate faster scan speed, but decreased MS2 identification rate. On the contrary, increasing the injection time of MS2 scan could increase identification rate but decrease scan speed and the number of identified MS2 spectra. Dynamic exclusion time should be set properly according to the chromatography peak width. Furthermore, we found that the Orbitrap analyzer, rather than the analytical column, was easily saturated with higher loading amount, thus limited the dynamic range of MS1-based quantification. By using optimized parameters, 10 000 proteins and 110 000 unique peptides were identified by using 20 h of effective liquid chromatography (LC) gradient time. The study therefore illustrated the importance of synchronizing LC-MS precursor ion targeting, fragment ion detection, and chromatographic separation for high efficient data-dependent proteomics.

[more...]

Activation of the CARD8 inflammasome requires a disordered region

Cell reports. 2020. Chui, AJ et al. Mem Sloan Kettering Canc Ctr, Triinst PhD Program Chem Biol, New York, NY 10065 USA.

ABSTRACT: Several cytosolic pattern-recognition receptors (PRRs) form multiprotein complexes called canonical inflammasomes in response to intracellular danger signals. Canonical inflammasomes recruit and activate caspase-1 (CASP1), which in turn cleaves and activates inflammatory cytokines and gasdermin D (GSDMD), inducing pyroptotic cell death. Inhibitors of the dipeptidyl peptidases DPP8 and DPP9 (DPP8/9) activate both the human NLRP1 and CARD8 inflammasomes. NLRP1 and CARD8 have different N-terminal regions but have similar C-terminal regions that undergo autoproteolysis to generate two non-covalently associated fragments.

Here, we show that DPP8/9 inhibition activates a proteasomal degradation pathway that targets disordered and misfolded proteins for destruction. CARD8's N terminus contains a disordered region of similar to 160 amino acids that is recognized and destroyed by this degradation pathway, thereby freeing its C-terminal fragment to activate CASP1 and induce pyroptosis. Thus, CARD8 serves as an alarm to signal the activation of a degradation pathway for disordered and misfolded proteins.

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TransCirc: an interactive database for translatable circular RNAs based on multi-omics evidence

Nucleic Acids Research. 2020. Huang, WD et al. Chinese Acad Sci, Biomed Big Data Ctr, CAS MPG Partner Inst Computat Biol, CAS Key Lab Computat Biol,Shanghai Inst Nutr & Hl, Shanghai 200031, Peoples R China.

ABSTRACT: TransCirc (https://www.biosino.org/transcirc/) is a specialized database that provide comprehensive evidences supporting the translation potential of circular RNAs (circRNAs). This database was generated by integrating various direct and indirect evidences to predict coding potential of each human circRNA and the putative translation products. Seven types of evidences for circRNA translation were included: (i) ribosome/polysome binding evidences supporting the occupancy of ribosomes onto circRNAs; (ii) experimentally mapped translation initiation sites on circRNAs; (iii) internal ribosome entry site on circRNAs; (iv) published N-6-methyladenosine modification data in circRNA that promote translation initiation; (v) lengths of the circRNA specific open reading frames; (vi) sequence composition scores froma machine learning prediction of all potential open reading frames; (vii) mass spectrometry data that directly support the circRNA encoded peptides across back-splice junctions.

TransCirc provides a user-friendly searching/browsing interface and independent lines of evidences to predicte how likely a circRNA can be translated. In addition, several flexible tools have been developed to aid retrieval and analysis of the data. TransCirc can serve as an important resource for investigating the translation capacity of circRNAs and the potential circRNA-encoded peptides, and can be expanded to include new evidences or additional species in the future.

[more...]

Protocol for Proximity-Dependent Proteomic Profiling in Yeast Cells by APEX and Alk-Ph Probe

STAR Protocols. 2020. Li, Yi et al. Peking University

ABSTRACT: Alk-Ph is a clickable APEX2 substrate developed for spatially restricted protein/RNA labeling in intact yeast cells. Alk-Ph is more water soluble and cell wall permeable than biotin-phenol substrate, allowing more efficient profiling of the subcellular proteome in microorganisms. We describe the protocol for Alk-Ph probe synthesis, APEX2 expression, and protein/RNA labeling in yeast and the workflow for quantitative proteomic experiments and data analysis. Using the yeast mitochondria as an example, we provide guidelines to achieve high-resolution mapping of subcellular yeast proteome and transcriptome.

For complete details on the use and execution of this protocol, please refer to Li etal. (2020). © 2020 The Author(s).

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Comprehensive identification of native medium-sized and short bioactive peptides in sea bass muscle

Food Chemistry. 2020. Cerrato, A et al. Sapienza Univ Roma, Dept Chem, Piazzale Aldo Moro 5, I-00185 Rome, Italy.

ABSTRACT: Native peptides from sea bass muscle were analyzed by two different approaches: medium-sized peptides by peptidomics analysis, whereas short peptides by suspect screening analysis employing an inclusion list of exact m/z values of all possible amino acid combinations (from 2 up to 4). The method was also extended to common post-translational modifications potentially interesting in food analysis, as well as non-proteolytic aminoacyl derivatives, which are well-known taste-active building blocks in pseudo-peptides.

The medium-sized peptides were identified by de novo and combination of de novo and spectra matching to a protein sequence database, with up to 4077 peptides (2725 modified) identified by database search and 2665 peptides (223 modified) identified by de novo only; 102 short peptide sequences were identified (with 12 modified ones), and most of them had multiple reported bioactivities. The method can be extended to any peptide mixture, either endogenous or by protein hydrolysis, from other food matrices.

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Comprehensive structural glycomic characterization of the glycocalyxes of cells and tissues

NATURE PROTOCOLS. 2020. Li, QY et al. Univ Calif Davis, Dept Chem, Davis, CA 95616 USA.

ABSTRACT: The glycocalyx comprises glycosylated proteins and lipids and fcorms the outermost layer of cells. It is involved in fundamental inter- and intracellular processes, including non-self-cell and self-cell recognition, cell signaling, cellular structure maintenance, and immune protection. Characterization of the glycocalyx is thus essential to understanding cell physiology and elucidating its role in promoting health and disease. This protocol describes how to comprehensively characterize the glycocalyx N-glycans and O-glycans of glycoproteins, as well as intact glycolipids in parallel, using the same enriched membrane fraction.

Profiling of the glycans and the glycolipids is performed using nanoflow liquid chromatography-mass spectrometry (nanoLC-MS). Sample preparation, quantitative LC-tandem MS (LC-MS/MS) analysis, and data processing methods are provided. In addition, we discuss glycoproteomic analysis that yields the site-specific glycosylation of membrane proteins. To reduce the amount of sample needed, N-glycan, O-glycan, and glycolipid analyses are performed on the same enriched fraction, whereas glycoproteomic analysis is performed on a separate enriched fraction. The sample preparation process takes 2-3 d, whereas the time spent on instrumental and data analyses could vary from 1 to 5 d for different sample sizes. This workflow is applicable to both cell and tissue samples. Systematic changes in the glycocalyx associated with specific glycoforms and glycoconjugates can be monitored with quantitation using this protocol. The ability to quantitate individual glycoforms and glycoconjugates will find utility in a broad range of fundamental and applied clinical studies, including glycan-based biomarker discovery and therapeutics. This protocol describes nanoflow liquid chromatography-mass spectrometry (nanoLC-MS) analysis of the N-glycans and O-glycans of glycoproteins and glycolipids, as well as site-specific glycosylation of membrane proteins.

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A quantitative thiol reactivity profiling platform to analyze redox and electrophile reactive cysteine proteomes

NATURE PROTOCOLS. 2020. Fu, L et al. Beijing Inst Life, State Key Lab Prote, Beijing Proteome Res Ctr, Natl Ctr Prot Sci Beijing, Beijing, Peoples R China.

ABSTRACT: Cysteine is unique among all protein-coding amino acids, owing to its intrinsically high nucleophilicity. The cysteinyl thiol group can be covalently modified by a broad range of redox mechanisms or by various electrophiles derived from exogenous or endogenous sources. Measuring the response of protein cysteines to redox perturbation or electrophiles is critical for understanding the underlying mechanisms involved. Activity-based protein profiling based on thiol-reactive probes has been the method of choice for such analyses.

We therefore adapted this approach and developed a new chemoproteomic platform, termed 'QTRP' (quantitative thiol reactivity profiling), that relies on the ability of a commercially available thiol-reactive probe IPM (2-iodo-N-(prop-2-yn-1-yl)acetamide) to covalently label, enrich and quantify the reactive cysteinome in cells and tissues. Here, we provide a detailed and updated workflow of QTRP that includes procedures for (i) labeling of the reactive cysteinome from cell or tissue samples (e.g., control versus treatment) with IPM, (ii) processing the protein samples into tryptic peptides and tagging the probe-modified peptides with isotopically labeled azido-biotin reagents containing a photo-cleavable linker via click chemistry reaction, (iii) capturing biotin-conjugated peptides with streptavidin beads, (iv) identifying and quantifying the photo-released peptides by mass spectrometry (MS)-based shotgun proteomics and (v) interpreting MS data by a streamlined informatic pipeline using a proteomics software, pFind 3, and an automatic post-processing algorithm. We also exemplified here how to use QTRP for mining H2O2-sensitive cysteines and for determining the intrinsic reactivity of cysteines in a complex proteome. We anticipate that this protocol should find broad applications in redox biology, chemical biology and the pharmaceutical industry. The protocol for sample preparation takes 3 d, whereas MS measurements and data analyses require 75 min and <30 min, respectively, per sample. Proteomic cysteines can undergo redox reactions and electrophile-derived modifications. In QTRP, a thiol-reactive probe is used to covalently label, enrich and quantify the reactive cysteinome in cultured cells and tissue samples.

[more...]

鸽毛滴虫外泌体的分离鉴定及蛋白质谱分析

Acta Veterinaria et Zootechnica Sinica (畜牧兽医学报). 2020. Ni, xinai et al. 中国农业科学院北京畜牧兽医研究所

ABSTRACT: The aim of this study was to determine whether Trichomonas gallinae(T.gallinae)secrete exosomes and investigate the protein composition of exosomes derived fromT.gallinae.T.gallinae was isolated from upper digestive tract of infected pigeons and identified by microscopy and sequencing.Exosomes were extracted from serum absent culture medium,and further verified by transmission electron microscopy,Western blot and nanoparticle tracking analysis(NTA).Label-free was used to identify the proteins of exosomes.Results showed that the parasite had T.gallinae morphology,and sequence alignment of ITS1/5.8S/ITS2gene showed an identity of 98%.The isolated vesicles presented a typical cup-shaped morphology;NTA results showed that particle size concentrated on 125.1nm,percentage of peak diameter at 132.3nm was 99.3%;proteins such as CD63and TSG101were conspicuously detected,suggesting that the extracts were exosomes.Mass spectrometry results showed that enolase,glyceraldehyde-3-phosphate dehydrogenase,phosphoglycerate mutase and elongation factor were the highly expressed proteins.GO pathways showed that proteins of exosomes mainly enriched into cellular components such as intracellular and cytoplasmic,played GTP binding and GTPase activity function, and participated in biological process of small GTPase mediated signal transduction and glycolytic process.KEGG enrichment analysis showed that proteins of exosomes were enriched in pathways as metabolic pathways,glycolysis/gluconeogenesis,biosynthesis of antibiotics and secondary metabolites.These results indicated that T.gallinaecan secrete exosomes and proteins of exosomes might play roles in energy metabolism,signal transduction and biosynthesis.

Combination of continuous digestion by peptidase and spectral similarity comparisons for peptide sequencing

JOURNAL OF SEPARATION SCIENCE. 2020. Yang, C et al. Chinese Acad Sci, Natl Chromatog Res & Anal Ctr, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, 457 Zhongshan Rd, Dalian 116023, Peoples R China.

ABSTRACT: Peptide sequencing is critical to the quality control of peptide drugs and functional studies of active peptides. A combination of peptidase digestion and mass spectrometry technology is common for peptide sequencing. However, such methods often cannot obtain the complete sequence of a peptide due to insufficient amino acid sequence information. Here, we developed a method of generating full peptide ladders and comparing their MS(2)spectral similarities. The peptide ladders, of which each component was different from the next component with one residue, were generated by continuous digestion by peptidase (carboxypeptidase Y and aminopeptidase).

Then, based on the characteristics of peptide ladders, complete sequencing was realized by comparing MS(2)spectral similarity of the generated peptide ladders. The complete amino acid sequences of bivalirudin, adrenocorticotropic hormone, and oxytocin were determined with high accuracy. This approach is beneficial to the quality control of drug peptides as well as the identification of novel bioactive peptides.

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Identification of modified peptides using localization-aware open search

Nature communications. 2020. Yu, FC et al. Univ Michigan, Dept Pathol, Ann Arbor, MI 48109 USA.

ABSTRACT: Identification of post-translationally or chemically modified peptides in mass spectrometry-based proteomics experiments is a crucial yet challenging task. We have recently introduced a fragment ion indexing method and the MSFragger search engine to empower an open search strategy for comprehensive analysis of modified peptides. However, this strategy does not consider fragment ions shifted by unknown modifications, preventing modification localization and limiting the sensitivity of the search. Here we present a localization-aware open search method, in which both modification-containing (shifted) and regular fragment ions are indexed and used in scoring.

We also implement a fast mass calibration and optimization method, allowing optimization of the mass tolerances and other key search parameters. We demonstrate that MSFragger with mass calibration and localization-aware open search identifies modified peptides with significantly higher sensitivity and accuracy. Comparing MSFragger to other modification-focused tools (pFind3, MetaMorpheus, and TagGraph) shows that MSFragger remains an excellent option for fast, comprehensive, and sensitive searches for modified peptides in shotgun proteomics data. Mass spectrometry-based proteomics is the method of choice for the global mapping of post-translational modifications, but matching and scoring peaks with unknown masses remains challenging. Here, the authors present a refined open search strategy to score all peaks with higher sensitivity and accuracy.

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Rapid and easy enrichment strategy for naturally acetylated N termini based on LysN digestion and amine-reactive resin capture

Analytical chemistry. 2020. Du, XX et al. Fudan Univ, Shanghai Canc Ctr, Shanghai 200032, Peoples R China.

ABSTRACT: Protein N-terminal acetylation (N-alpha-acetylation) is one of the most common modifications in both eukaryotes and prokaryotes. Although studies have shown that N-alpha-acetylation plays important roles in protein assembly, stability, and location, the physiological role has not been fully elucidated. Therefore, a robust and large-scale analytical method is important for a better understanding of N-alpha-acetylation. Here, an enrichment strategy was presented based on LysN digestion and amine-reactive resin capture to study naturally acetylated protein N termini.

Since LysN protease cleaves at the amino-terminus of the lysine residue, all resulting peptides except naturally acetylated N-terminal peptides contain free amino groups and can be removed by coupling with AminoLink Resin. Therefore, the naturally acetylated N-terminal peptides were left in solution and enriched for further liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The method was very simple and fast, which contained no additional chemical derivatization except protein reduction and alkylation necessarily needed in bottom-up proteomics. It could be used to study acetylated N termini from complex biological samples without bias toward different peptides with various physicochemical properties. The enrichment specificity was above 99% when it was applied in HeLa cell lysates. Neo-N termini generated by endogenous degradation could be directly distinguished without the use of stable-isotope labeling because no chemical derivatization was introduced in this method. Furthermore, this method was highly complementary to the traditional analytical methods for protein N termini based on trypsin only with ArgC-like activity. Therefore, the described method was beneficial to naturally acetylated protein N termini profiling.

[more...]

Investigation of indigoidine synthetase reveals a conserved active-site base residue of nonribosomal peptide synthetase oxidases

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. 2020. Pang, B et al. Univ Calif Berkeley, QB3 Inst, Berkeley, CA 94720 USA.

ABSTRACT: Nonribosomal peptide synthetase (NRPS) oxidase (Ox) domains oxidize protein-bound intermediates to install crucial structural motifs in bioactive natural products. The mechanism of this domain remains elusive. Here, by studying indigoidine synthetase, a single-module NRPS involved in the biosynthesis of indigoidine and several other bacterial secondary metabolites, we demonstrate that its Ox domain utilizes an active-site base residue, tyrosine 665, to deprotonate a protein-bound L-glutaminyl residue.

We further validate the generality of this active-site residue among NRPS Ox domains. These findings not only resolve the biosynthetic pathway mediated by indigoidine synthetase but enable mechanistic insight into NRPS Ox domains.

[more...]

The proteome landscape of the kingdoms of life

Nature. 2020. Muller, JB et al. Max Planck Inst Biochem, Dept Prote & Signal Transduct, Martinsried, Germany.

ABSTRACT: Proteins carry out the vast majority of functions in all biological domains, but for technological reasons their large-scale investigation has lagged behind the study of genomes. Since the first essentially complete eukaryotic proteome was reported(1), advances in mass-spectrometry-based proteomics(2)have enabled increasingly comprehensive identification and quantification of the human proteome(3-6). However, there have been few comparisons across species(7,8), in stark contrast with genomics initiatives(9).

Here we use an advanced proteomics workflow-in which the peptide separation step is performed by a microstructured and extremely reproducible chromatographic system-for the in-depth study of 100 taxonomically diverse organisms. With two million peptide and 340,000 stringent protein identifications obtained in a standardized manner, we double the number of proteins with solid experimental evidence known to the scientific community. The data also provide a large-scale case study for sequence-based machine learning, as we demonstrate by experimentally confirming the predicted properties of peptides fromBacteroides uniformis. Our results offer a comparative view of the functional organization of organisms across the entire evolutionary range. A remarkably high fraction of the total proteome mass in all kingdoms is dedicated to protein homeostasis and folding, highlighting the biological challenge of maintaining protein structure in all branches of life. Likewise, a universally high fraction is involved in supplying energy resources, although these pathways range from photosynthesis through iron sulfur metabolism to carbohydrate metabolism. Generally, however, proteins and proteomes are remarkably diverse between organisms, and they can readily be explored and functionally compared at www.proteomesoflife.org.

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A comprehensive evaluation of MS/MS spectrum prediction tools for shotgun proteomics

Proteomics. 2020. Xu, R et al. Beijing Inst Life, Natl Ctr Prot Sci Beijing, Beijing Proteome Res Ctr, State Key Lab Prote, Beijing 102206, Peoples R China.

ABSTRACT: Spectrum prediction using machine learning or deep learning models is an emerging method in computational proteomics. Several deep learning-based MS/MS spectrum prediction tools have been developed and showed their potentials not only for increasing the sensitivity and accuracy of data-dependent acquisition search engines, but also for building spectral libraries for data-independent acquisition analysis. Different tools with their unique algorithms and implementations may result in different performances.

Hence, it is necessary to systematically evaluate these tools to find out their preferences and intrinsic differences. In this study, multiple datasets with different collision energies, enzymes, instruments, and species, are used to evaluate the performances of the deep learning-based MS/MS spectrum prediction tools, as well as, the machine learning-based tool MS2PIP. The evaluations may provide helpful insights and guidelines of spectrum prediction tools for the corresponding researchers.

[more...]

A clickable APEX probe for proximity-dependent proteomic profiling in yeast

Cell Chemical Biology. 2020. Li, Y et al. Peking Univ, Coll Chem & Mol Engn, Key Lab Bioorgan Chem & Mol Engn,Minist Educ, Beijing Natl Lab Mol Sci,Synthet & Funct Biomol C, Beijing 100871, Peoples R China.

ABSTRACT: The engineered ascorbate peroxidase (APEX) is a powerful tool for the proximity-dependent labeling of proteins and RNAs in live cells. Although widely use in mammalian cells, APEX applications in microorganisms have been hampered by the poor labeling efficiency of its biotin-phenol (BP) substrate. In this study, we sought to address this challenge by designing and screening a panel of alkyne-functionalized substrates. Our best probe, Alk-Ph, substantially improves APEX-labeling efficiency in intact yeast cells, as it is more cell wall-permeant than BP.

Through a combination of protein-centric and peptide-centric chemoproteomic experiments, we have identified 165 proteins with a specificity of 94% in the yeast mitochondrial matrix. In addition, we have demonstrated that Alk-Ph is useful for proximity-dependent RNA labeling in yeast, thus expanding the scope of APEX-seq. We envision that this improved APEX-labeling strategy would set the stage for the large-scale mapping of spatial proteome and transcriptome in yeast.

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Proteomic Analysis Reveals that EPHX1 Contributes to 5-Fluorouracil Resistance in a Human Hepatocellular Carcinoma Cell Line

Proteomics Clinical Applications. 2020. Sun, R et al. Dalian Med Univ, Dept Gen Surg, Div Hepatobiliary & Pancreat Surg, Affiliated Hosp 2, Dalian 116023, Peoples R China.

ABSTRACT: Purpose The extensive drug resistance of hepatocellular carcinoma (HCC) has become a major cause of chemotherapy failure. A deeper understanding of the drug resistance mechanism of tumor cells is very significant for improving the clinical prognosis of patients with HCC. Experimental Design In this study, proteomic studies on the composition of 5-fluorouracil (5-Fu) resistant Bel/5Fu cell line and its parent Bel7402 cell line by using an ionic liquid assisted proteins extraction method with the advantage of extracting plasma membrane proteins to a wider extent are performed.

Then the expression level and function of differentially expressed plasma membrane proteins are verified. Results In total, 25 plasma membrane proteins are shown differentially expressed in Bel/5Fu compared with Bel7402. Western blot analysis results further confirmed that the EPHX1 PLIN2 RAB27B SLC4A2 are upregulated in Bel/5Fu cells in accordance with the proteomics data. Moreover, cell viability assay and clonogenic survival assay results demonstrated that EPHX1 is closely related to the chemoresistance of Bel/5Fu to 5-Fu. Conclusions and Clinical Relevance Plasma membrane protein EPHX1 is closely related to the chemotherapy resistance of Bel/5Fu cells and can be used as a new drug target to improve the clinical prognosis of patients with HCC.

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Identification of novel DPP--IV inhibitory peptides from Atlantic salmon (Salmo salar) skin

Food Research International. 2020. Jin, RT et al. Northeast Agr Univ, Coll Food Sci, 600 Changjiang Rd, Harbin 150030, Peoples R China.

ABSTRACT: The aim of this study was to identify dipeptidyl peptidase IV (DPP-IV) inhibitory peptides from salmon skin collagen hydrolysate, and to evaluate the possible inhibition mechanism of DPP-IV and peptide. Salmon skin collagen was hydrolyzed by pepsin, trypsin, papain, or Alcalase 2.4 L, separately. Trypsin hydrolysate (10 mg/mL) showed the highest inhibitory activity of 66.12 +/- 0.68%. The hydrolysate was separated into three fractions by ultrafiltration, and the inhibitory IC50 of M1 (molecular weight< 3 kDa) was 1.54 +/- 0.06 mg/mL.

M1 was separated by gel chromatography and RP-HPLC; A10 was the highest inhibitory fraction in the 12 fractions, i.e., IC50 was 0.79 +/- 0.13 mg/mL. A novel peptide LDKVFR with the IC50 value of 0.1 +/- 0.03 mg/mL (128.71 mu M) was identified from A10. Molecular docking revealed that six hydrogen bonds and eight hydrophobic interactions between LDKVFR and DPP-IV were contributed to DPP-IV inhibition.

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Sequence analysis of digestion-resistant peptides may be an efficient strategy for studying the linear epitopes of Jug r 1, the major walnut allergen

Food Chemistry. 2020. Guo, XY et al. China Agr Univ, Coll Food Sci & Nutr Engn, Beijing Adv Innovat Ctr Food Nutr & Human Hlth, 17 Qinghua East Rd, Beijing, Peoples R China.

ABSTRACT: Jug r 1, the major allergen of walnut, triggers severe allergic reactions through epitopes. Hence, research on the efficient strategy for analyzing the linear epitopes of Jug r 1 are necessary. In this work, bioinformatics analysis was used to predict the linear epitopes of Jug r 1. Overlapping peptide synthesis was used to map linear epitopes. In vitro simulated gastrointestinal digestion and HPLC-MS/MS were used to identify digestion-resistant peptides. The results showed that six predicted linear epitopes were AA28-35, AA42-49, AA55-62, AA65-73, AA97-104, and AA109-121.

AA16-30 and AA125-139 were identified by the sera of walnut allergic patients. Five digestionresistant peptides were AA19-33, AA40-45, AA54-74, AA96-106, and AA117-137. The predicted results only included one of the linear epitopes identified by sera, while the digestion-resistant peptides covered all. Therefore, the digestion-resistant property of food allergens may be a promising direction for studying the linear epitopes of Jug r 1.

[more...]

Characterization of urinary exosomes purified with size exclusion chromatography and ultracentrifugation

Journal of Proteome Research. 2020. Guan, S et al. Fudan Univ, Dept Chem, Shanghai 200438, Peoples R China.

ABSTRACT: Exosomes, a subtype of extracellular vesicles secreted by mammalian cells with a typical size range of 30-150 nm, have been implicated in many biological processes as intercellular communication carriers. The isolation of exosomes is an essential and challenging step before subsequent analysis and functional studies, due to the complexity of body fluids, as well as the small size and low density of exosomes. Ultracentrifugation (UC) and size exclusion chromatography (SEC) are two methods that have been extensively used for exosomes isolation in biological studies in recent years.

In this work, we compared the characteristics of urinary exosomes extracted with SEC and UC methods in detail. Results showed that the SEC isolation method was superior to UC in the recovery of exosomal particles and proteins. The results of proteomics analysis showed that more purified exosomes were extracted with the SEC method. We also observed that parts of exosomes were ruptured and precipitated insufficiently during UC isolations. It not only led to a low recovery of exosome proteins but also resulted in a considerable loss of exosomal particles. Moreover, the exosomal rupture and particle loss in UC could not be avoided by resuspension of the exosomal particles. Our results also showed that exosomes from SEC purifications possessed a high internalization capability from 4 to 6 h when incubated with EA.hy926 and HCV29 cell lines.

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An Arabidopsis secondary metabolite directly targets expression of the bacterial type III secretion system to inhibit bacterial virulence

Cell host & microbe. 2020. Wang, W et al. Chinese Acad Sci, Innovat Acad Seed Design, Inst Genet & Dev Biol, State Key Lab Plant Genom, Beijing 100101, Peoples R China.

ABSTRACT: Plants deploy a variety of secondary metabolites to fend off pathogen attack. Although defense compounds are generally considered toxic to microbes, the exact mechanisms are often unknown. Here, we show that the Arabidopsis defense compound sulforaphane (SFN) functions primarily by inhibiting Pseudomonas syringae type III secretion system (TTSS) genes, which are essential for pathogenesis. Plants lacking the aliphatic glucosinolate pathway, which do not accumulate SFN, were unable to attenuate TTSS gene expression and exhibited increased susceptibility to P.

syringae strains that cannot detoxify SFN. Chemoproteomics analyses showed that SFN covalently modified the cysteine at position 209 of HrpS, a key transcription factor controlling TTSS gene expression. Site-directed mutagenesis and functional analyses further confirmed that Cys209 was responsible for bacterial sensitivity to SFN in vitro and sensitivity to plant defenses conferred by the aliphatic glucosinolate pathway. Collectively, these results illustrate a previously unknown mechanism by which plants disarma pathogenic bacterium.

[more...]

Parallel channels-multidimensional protein identification technology

Journal of the American Society for Mass Spectrometry. 2020. Zhang, N et al. Peking Univ, Sch Pharmaceut Sci, Sch Basic Med Sci, Hlth Sci Ctr,Ctr Precis Med Multi Res, Beijing 100191, Peoples R China.

ABSTRACT: Multidimensional protein identification (MudPIT), developed in the Yates Laboratory 20 years ago, is regarded as a powerful tool for proteomics research. Due to its remarkable online separation advantages, MudPIT has been widely used to facilitate discoveries in the field of proteomics research. However, it has one major disadvantage: the process of eluting peptides during strong cation exchange introduces salts, of different concentrations, into the mass spectrometer. Considering the sensitivity of the new generation of high-resolution mass spectrometers, developing a new version of MudPIT that could eliminate the introduction of salts in the elute would be a significant advancement to current technology.

Herein, we developed a new, clean version of MudPIT called parallel channels-multidimensional protein identification technology (PC-MudPIT) to overcome this issue. In this design, the original biphasic trapping column was replaced by two parallel analytical column channels. We successfully averted the salt contamination yet retained all the other advantages of MudPIT. A total of 8161 and 7359 protein groups were identified from A549 whole cell lysate using PC-MudPIT and classic MudPIT, respectively. Moreover, we discovered the additional advantage that, in online mode, PC-MudPIT can also be used for an enrichment process of phosphopeptide identification. We identified a total 11453 phosphopeptides using PC-MudPIT and 7729 phosphopeptides using offline TiO2 enrichment followed by classic MudPIT. These advances indicate the possibility of other innovative applications of PC-MudPIT technology in deep proteome exploration.

[more...]

Sequential amidation of peptide C-termini for improving fragmentation efficiency

Journal of Mass Spectrometry. 2020. Wu, Q et al. Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog Res & Anal Ctr, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China.

ABSTRACT: Owing to the poor fragmentation efficiency caused by the lack of a positively charged basic group at the C-termini of peptides, the identification of nontryptic peptides in classical proteomics is known to be less efficient. Particularly, attaching positively charged basic groups to C-termini via chemical derivatizations is known to be able to enhance their fragmentation efficiency. In this study, we introduced a novel strategy, C-termini sequential amidation reaction (CSAR), to improve peptide fragmentation efficiency.

By this strategy, C-terminal and side-chain carboxyl groups were firstly amidated by neutral methylamine (MA), and then C-terminal amide bonds were selectively deamidated through peptide amidase while side-chain amide bonds remained unchanged, followed by the secondary amidation of C-termini via basic agmatine (AG). We optimized the amidation reaction conditions to achieve the MA derivatization efficiency of >99% for side-chain carboxyl groups and AG derivatization efficiency of 80% for the hydrolytic C-termini. We applied CSAR strategy to identify bovine serum albumin (BSA) chymotryptic digests, resulting in the increased fragmentation efficiencies (improvement by 9-32%) and charge states (improvement by 39-52%) under single or multiple dissociation modes. The strategy described here might be a promising approach for the identification of peptides that suffered from poor fragmentation efficiency.

[more...]

The F1 loop of the talin head domain acts as a gatekeeper in integrin activation and clustering

Journal of cell science. 2020. Kukkurainen, S et al. Tampere Univ, Fac Med & Hlth Technol, Arvo Ylpon Katu 34, FI-33520 Tampere, Finland.

ABSTRACT: Integrin activation and clustering by talin are early steps of cell adhesion. Membrane-bound talin head domain and kindlin bind to the beta integrin cytoplasmic tail, cooperating to activate the heterodimeric integrin, and the talin head domain induces integrin clustering in the presence of Mn2+. Here we show that kindlin-1 can replace Mn2+ to mediate beta 3 integrin clustering induced by the talin head, but not that induced by the F2-F3 fragment of talin. Integrin clustering mediated by kindlin-1 and the talin head was lost upon deletion of the flexible loop within the talin head F1 subdomain.

Further mutagenesis identified hydrophobic and acidic motifs in the F1 loop responsible for beta 3 integrin clustering. Modeling, computational and cysteine crosslinking studies showed direct and catalytic interactions of the acidic F1 loop motif with the juxtamembrane domains of alpha- and beta 3-integrins, in order to activate the beta 3 integrin heterodimer, further detailing the mechanism by which the talin-kindlin complex activates and clusters integrins. Moreover, the F1 loop interaction with the beta 3 integrin tail required the newly identified compact FERM fold of the talin head, which positions the F1 loop next to the inner membrane clasp of the talin-bound integrin heterodimer. This article has an associated First Person interview with the first author of the paper.

[more...]

Structure of nucleosome-bound human BAF complex

SCIENCE. 2020. He, S et al. Fudan Univ, Shanghai Med Coll, State Key Lab Genet Engn, Shanghai Canc Ctr,Inst Biomed Sci, Shanghai 200032, Peoples R China.

ABSTRACT: Mammalian SWI/SNF family chromatin remodelers, BRG1/BRM-associated factor (BAF) and polybromoassociated BAF (PBAF), regulate chromatin structure and transcription, and their mutations are linked to cancers. The 3.7-angstrom-resolution cryo-electron microscopy structure of human BAF bound to the nucleosome reveals that the nucleosome is sandwiched by the base and the adenosine triphosphatase (ATPase) modules, which are bridged by the actin-related protein (ARP) module. The ATPase motor is positioned proximal to nucleosomal DNA and, upon ATP hydrolysis, engages with and pumps DNA along the nucleosome.

The C-terminal alpha helix of SMARCB1, enriched in positively charged residues frequently mutated in cancers, mediates interactions with an acidic patch of the nucleosome. AT-rich interactive domain-containing protein 1A (ARID1A) and the SWI/SNF complex subunit SMARCC serve as a structural core and scaffold in the base module organization, respectively. Our study provides structural insights into subunit organization and nucleosome recognition of human BAF complex.

[more...]

High-density chemical cross-linking for modeling protein interactions

PNAS. 2020. Mintseris, J et al. Harvard Med Sch, Dept Cell Biol, Boston, MA 02115 USA.

ABSTRACT: Detailed mechanistic understanding of protein complex function is greatly enhanced by insights from its 3-dimensional structure. Traditional methods of protein structure elucidation remain expensive and labor-intensive and require highly purified starting material. Chemical cross-linking coupled with mass spectrometry offers an alternative that has seen increased use, especially in combination with other experimental approaches like cryo-electron microscopy. Here we report advances in method development, combining several orthogonal cross-linking chemistries as well as improvements in search algorithms, statistical analysis, and computational cost to achieve coverage of 1 unique cross-linked position pair for every 7 amino acids at a 1% false discovery rate.

This is accomplished without any peptide-level fractionation or enrichment. We apply our methods to model the complex between a carbonic anhydrase (CA) and its protein inhibitor, showing that the cross-links are selfconsistent and define the interaction interface at high resolution. The resulting model suggests a scaffold for development of a class of protein-based inhibitors of the CA family of enzymes. We next cross-link the yeast proteasome, identifying 3,893 unique crosslinked peptides in 3 mass spectrometry runs. The dataset includes 1,704 unique cross-linked position pairs for the proteasome subunits, more than half of them intersubunit. Using multiple recently solved cryo-EM structures, we show that observed cross-links reflect the conformational dynamics and disorder of some proteasome subunits. We further demonstrate that this level of cross-linking density is sufficient to model the architecture of the 19-subunit regulatory particle de novo.

[more...]

Nicotinamide mononucleotide adenylyltransferase uses its NAD+ substrate-binding site to chaperone phosphorylated Tau

eLife. 2020. Ma, XJ et al. Chinese Acad Sci, Interdisciplinary Res Ctr Biol & Chem, Shanghai Inst Organ Chem, Shanghai, Peoples R China.

ABSTRACT: Tau hyper-phosphorylation and deposition into neurofibrillary tangles have been found in brains of patients with Alzheimer's disease (AD) and other tauopathies. Molecular chaperones are involved in regulating the pathological aggregation of phosphorylated Tau (pTau) and modulating disease progression. Here, we report that nicotinamide mononucleotide adenylyltransferase (NMNAT), a well-known NAD(+) synthase, serves as a chaperone of pTau to prevent its amyloid aggregation in vitro as well as mitigate its pathology in a fly tauopathy model.

By combining NMR spectroscopy, crystallography, single-molecule and computational approaches, we revealed that NMNAT adopts its enzymatic pocket to specifically bind the phosphorylated sites of pTau, which can be competitively disrupted by the enzymatic substrates of NMNAT. Moreover, we found that NMNAT serves as a co-chaperone of Hsp90 for the specific recognition of pTau over Tau. Our work uncovers a dedicated chaperone of pTau and suggests NMNAT as a key node between NAD+ metabolism and Tau homeostasis in aging and neurodegeneration.

[more...]

A molecular switch regulating transcriptional repression and activation of PPARγ

Nature communications. 2020. Shang, JS et al. Scripps Res Inst, Dept Integrat Struct & Computat Biol, Jupiter, FL 33458 USA.

ABSTRACT: Nuclear receptor (NR) transcription factors use a conserved activation function-2 (AF-2) helix 12 mechanism for agonist-induced coactivator interaction and NR transcriptional activation. In contrast, ligand-induced corepressor-dependent NR repression appears to occur through structurally diverse mechanisms. We report two crystal structures of peroxisome proliferator-activated receptor gamma (PPAR gamma) in an inverse agonist/corepressor-bound transcriptionally repressive conformation. Helix 12 is displaced from the solvent-exposed active conformation and occupies the orthosteric ligand-binding pocket enabled by a conformational change that doubles the pocket volume.

Paramagnetic relaxation enhancement (PRE) NMR and chemical crosslinking mass spectrometry confirm the repressive helix 12 conformation. PRE NMR also defines the mechanism of action of the corepressor-selective inverse agonist T0070907, and reveals that apo-helix 12 exchanges between transcriptionally active and repressive conformations-supporting a fundamental hypothesis in the NR field that helix 12 exchanges between transcriptionally active and repressive conformations. Structural studies of nuclear receptor transcription factors revealed that nearly all nuclear receptors share a conserved helix 12 dependent transcriptional activation mechanism. Here the authors present two crystal structures of peroxisome proliferator-activated receptor gamma (PPAR gamma) in an inverse agonist/corepressor-bound transcriptionally repressive conformation, where helix 12 is located within the orthosteric ligand-binding pocket instead, and discuss mechanistic implications.

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Structures of three MORN repeat proteins and a re-evaluation of the proposed lipid-binding properties of MORN repeats

PLOS ONE. 2020. Sajko, S et al. Univ Vienna, Dept Struct & Computat Biol, Max Perutz Labs, Vienna, Austria.

ABSTRACT: MORN (Membrane Occupation and Recognition Nexus) repeat proteins have a wide taxonomic distribution, being found in both prokaryotes and eukaryotes. Despite this ubiquity, they remain poorly characterised at both a structural and a functional level compared to other common repeats. In functional terms, they are often assumed to be lipid-binding modules that mediate membrane targeting. We addressed this putative activity by focusing on a protein composed solely of MORN repeats-Trypanosoma brucei MORN1.

Surprisingly, no evidence for binding to membranes or lipid vesicles by TbMORN1 could be obtained either in vivo or in vitro. Conversely, TbMORN1 did interact with individual phospholipids. High- and low-resolution structures of the MORN1 protein from Trypanosoma brucei and homologous proteins from the parasites Toxoplasma gondii and Plasmodium falciparum were obtained using a combination of macromolecular crystallography, small-angle X-ray scattering, and electron microscopy. This enabled a first structure-based definition of the MORN repeat itself. Furthermore, all three structures dimerised via their C-termini in an antiparallel configuration. The dimers could form extended or V-shaped quaternary structures depending on the presence of specific interface residues. This work provides a new perspective on MORN repeats, showing that they are protein-protein interaction modules capable of mediating both dimerisation and oligomerisation.

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The Nucleosome Remodeling and Deacetylase complex has an asymmetric, dynamic, and modular architecture

Cell reports. 2020. Low, JKK et al. Univ Sydney, Sch Life & Environm Sci, Sydney, NSW, Australia.

ABSTRACT: The nucleosome remodeling and deacetylase (NuRD) complex is essential for metazoan development but has been refractory to biochemical analysis. We present an integrated analysis of the native mammalian NuRD complex, combining quantitative mass spectrometry, cross-linking, protein biochemistry, and electron microscopy to define the architecture of the complex. NuRD is built from a 2:2:4 (MTA, HDAC, and RBBP) deacetylase module and a 1:1:1 (MBD, GATAD2, and Chromodomain-Helicase-DNA-binding [CHD]) remodeling module, and the complex displays considerable structural dynamics.

The enigmatic GATAD2 controls the asymmetry of the complex and directly recruits the CHD remodeler. The MTA-MBD interaction acts as a point of functional switching, with the transcriptional regulator PWWP2A competing with MBD for binding to the MTA-HDAC-RBBP subcomplex. Overall, our data address the long-running controversy over NuRD stoichiometry, provide imaging of the mammalian NuRD complex, and establish the biochemical mechanism by which PWWP2A can regulate NuRD composition.

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Exaptation of two ancient immune proteins into a new dimeric pore-forming toxin in snails

JOURNAL OF STRUCTURAL BIOLOGY. 2020. Giglio, ML et al. Consejo Nacl Invest Cient & Tecn, Fdn Inst Leloir, IIBBA, Av Patricias Argentinas 435,C1405BWE, Buenos Aires, DF, Argentina.

ABSTRACT: The Membrane Attack Complex-Perforin (MACPF) family is ubiquitously found in all kingdoms. They have diverse cellular roles, however MACPFs with pore-forming toxic function in venoms and poisons are very rare in animals. Here we present the structure of PmPV2, a MACPF toxin from the poisonous apple snail eggs, that can affect the digestive and nervous systems of potential predators. We report the three-dimensional structure of PmPV2, at 17.2 angstrom resolution determined by negative-stain electron microscopy and its solution structure by small angle X-ray scattering (SAXS).

We found that PV2s differ from nearly all MACPFs in two respects: it is a dimer in solution and protomers combine two immune proteins into an AB toxin. The MACPF chain is linked by a single disulfide bond to a tachylectin chain, and two heterodimers are arranged head-to-tail by non-covalent forces in the native protein. MACPF domain is fused with a putative new Ct-accessory domain exclusive to invertebrates. The tachylectin is a six-bladed beta-propeller, similar to animal tectonins. We experimentally validated the predicted functions of both subunits and demonstrated for the first time that PV2s are true poreforming toxins. The tachylectin "B" delivery subunit would bind to target membranes, and then the MACPF "A" toxic subunit would disrupt lipid bilayers forming large pores altering the plasma membrane conductance. These results indicate that PV2s toxicity evolved by linking two immune proteins where their combined preexisting functions gave rise to a new toxic entity with a novel role in defense against predation. This structure is an unparalleled example of protein exaptation.

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Methionine sulfoxide reductase B from Corynebacterium diphtheriae catalyzes sulfoxide reduction via an intramolecular disulfide cascade

Journal of Biological Chemistry. 2020. Tossounian, MA et al. VUB, VIB, Ctr Structural Biol, B-1050 Brussels, Belgium.

ABSTRACT: Corynebacterium diphtheriae is a human pathogen that causes diphtheria. In response to immune system-induced oxidative stress, C. diphtheriae expresses antioxidant enzymes, among which are methionine sulfoxide reductase (Msr) enzymes, which are critical for bacterial survival in the face of oxidative stress. Although some aspects of the catalytic mechanism of the Msr enzymes have been reported, several details still await full elucidation. Here, we solved the solution structure of C. diphtheriae MsrB (Cd-MsrB) and unraveled its catalytic and oxidation-protection mechanisms.

Cd-MsrB catalyzes methionine sulfoxide reduction involving three redox-active cysteines. Using NMR heteronuclear single-quantum coherence spectra, kinetics, biochemical assays, and MS analyses, we show that the conserved nucleophilic residue Cys-122 is S-sulfenylated after substrate reduction, which is then resolved by a conserved cysteine, Cys-66, or by the nonconserved residue Cys-127. We noted that the overall structural changes during the disulfide cascade expose the Cys-122?Cys-66 disulfide to recycling through thioredoxin. In the presence of hydrogen peroxide, Cd-MsrB formed reversible intra- and intermolecular disulfides without losing its Cys-coordinated Zn2+, and only the nonconserved Cys-127 reacted with the low-molecular-weight (LMW) thiol mycothiol, protecting it from overoxidation. In summary, our structure-function analyses reveal critical details of the Cd-MsrB catalytic mechanism, including a major structural rearrangement that primes the Cys-122?Cys-66 disulfide for thioredoxin reduction and a reversible protection against excessive oxidation of the catalytic cysteines in Cd-MsrB through intra- and intermolecular disulfide formation and S-mycothiolation.

[more...]

crisscrosslinkeR: identification and visualization of protein--RNA and protein--protein interactions from crosslinking mass spectrometry

Bioinformatics. 2020. Gail, EH et al. Monash Univ, Dept Biochem & Mol Biol, Biomed Discovery Inst, Clayton, Vic 3800, Australia.

ABSTRACT: The Summary: Unbiased detection of protein-protein and protein-RNA interactions within ribonucleoprotein complexes are enabled through crosslinking followed by mass spectrometry. Yet, different methods detect different types of molecular interactions and therefore require the usage of different software packages with limited compatibility. We present crisscrosslinkeR, an R package that maps both protein-protein and protein-RNA interactions detected by different types of approaches for crosslinking with mass spectrometry.

crisscrosslinkeR produces output files that are compatible with visualization using popular software packages for the generation of publication-quality figures.

[more...]

A basic motif anchoring ISWI to nucleosome acidic patch regulates nucleosome spacing

Nature chemical biology. 2020. Dao, HT et al. Princeton Univ, Dept Chem, Princeton, NJ USA.

ABSTRACT: A photocrosslinking-based nucleosome profiling approach is used to identify a conserved basic motif in the ISWI remodeler SNF2h that anchors it to the acidic patch of nucleosome and enables nucleosome sliding activity. Recent studies have implicated the nucleosome acidic patch in the activity of ATP-dependent chromatin remodeling machines. We used a photocrosslinking-based nucleosome profiling technology (photoscanning) to identify a conserved basic motif within the catalytic subunit of ISWI remodelers, SNF2h, which engages this nucleosomal epitope.

This region of SNF2h is essential for chromatin remodeling activity in a reconstituted biochemical system and in cells. Our studies suggest that the basic motif in SNF2h plays a critical role in anchoring the remodeler to the nucleosomal surface. We also examine the functional consequences of several cancer-associated histone mutations that map to the nucleosome acidic patch. Kinetic studies using physiologically relevant heterotypic nucleosomal substrates ('Janus' nucleosomes) indicate that these cancer-associated mutations can disrupt regularly spaced chromatin structure by inducing ISWI-mediated unidirectional nucleosome sliding. These results indicate a potential mechanistic link between oncogenic histones and alterations to the chromatin landscape.

[more...]

ADAM17 cytoplasmic domain modulates Thioredoxin-1 conformation and activity

Redox biology. 2020. Costa, RAPE et al. CNPEM, Lab Nacl Biociencias, LNBio, Campinas, SP, Brazil.

ABSTRACT: The activity of Thioredoxin-1 (Trx-1) is adjusted by the balance of its monomeric, active and its dimeric, inactive state. The regulation of this balance is not completely understood. We have previously shown that the cytoplasmic domain of the transmembrane protein A Disintegrin And Metalloprotease 17 (ADAM17cyto) binds to Thioredoxin-1 (Trx-1) and the destabilization of this interaction favors the dimeric state of Trx-1. Here, we investigate whether ADAM17 plays a role in the conformation and activation of Trx-1.

We found that disrupting the interacting interface with Trx-1 by a site-directed mutagenesis in ADAM17 (ADAM17cyto(F730A)) caused a decrease of Trx-1 reductive capacity and activity. Moreover, we observed that ADAM17 overexpressing cells favor the monomeric state of Trx-1 while knockdown cells do not. As a result, there is a decrease of cell oxidant levels and ADAM17 sheddase activity and an increase in the reduced cysteine-containing peptides in intracellular proteins in ADAM17cyto overexpressing cells. A mechanistic explanation that ADAM17cyto favors the monomeric, active state of Trx-1 is the formation of a disulfide bond between Cys(824) at the C-terminal of ADAM17cyto with the Cys(73) of Trx-1, which is involved in the dimerization site of Trx-1. In summary, we propose that ADAM17 is able to modulate Trx-1 conformation affecting its activity and intracellular redox state, bringing up a novel possibility for positive regulation of thiol isomerase activity in the cell by mammalian metalloproteinases.

[more...]

A structural model of the endogenous human BAF complex informs disease mechanisms

Cell. 2020. Mashtalir, N et al. Dana Farber Canc Inst, Dept Pediat Oncol, Boston, MA 02115 USA.

ABSTRACT: Mammalian SWI/SNF complexes are ATP-dependent chromatin remodeling complexes that regulate genomic architecture. Here, we present a structural model of the endogenously purified human canonical BAF complex bound to the nucleosome, generated using cryoelectron microscopy (cryo-EM), cross-linking mass spectrometry, and homology modeling. BAF complexes bilaterally engage the nucleosome H2A/H2B acidic patch regions through the SMARCB1 C-terminal alpha-helix and the SMARCA4/2 C-terminal SnAc/post-SnAc regions, with disease-associated mutations in either causing attenuated chromatin remodeling activities.

Further, we define changes in BAF complex architecture upon nucleosome engagement and compare the structural model of endogenous BAF to those of related SWI/SNF-family complexes. Finally, we assign and experimentally interrogate cancer-associated hot-spot mutations localizing within the endogenous human BAF complex, identifying those that disrupt BAF subunit-subunit and subunit-nucleosome interfaces in the nucleosome-bound conformation. Taken together, this integrative structural approach provides important biophysical foundations for understanding the mechanisms of BAF complex function in normal and disease states.

[more...]

Structure of SWI/SNF chromatin remodeller RSC bound to a nucleosome

Nature. 2020. Wagner, FR et al. Max Planck Inst Biophys Chem, Dept Mol Biol, Gottingen, Germany.

ABSTRACT: The cryo-electron microscopy structure of the 16-subunit yeast SWI/SNF complex RSC in complex with a nucleosome substrate provides insights into the chromatin-remodelling function of this family of protein complexes. Chromatin-remodelling complexes of the SWI/SNF family function in the formation of nucleosome-depleted, transcriptionally active promoter regions (NDRs)(1,2). In the yeast Saccharomyces cerevisiae, the essential SWI/SNF complex RSC3 contains 16 subunits, including the ATP-dependent DNA translocase Sth1(4,5).

RSC removes nucleosomes from promoter regions(6,7) and positions the specialized +1 and -1 nucleosomes that flank NDRs(8,9). Here we present the cryo-electron microscopy structure of RSC in complex with a nucleosome substrate. The structure reveals that RSC forms five protein modules and suggests key features of the remodelling mechanism. The body module serves as a scaffold for the four flexible modules that we call DNA-interacting, ATPase, arm and actin-related protein (ARP) modules. The DNA-interacting module binds extra-nucleosomal DNA and is involved in the recognition of promoter DNA elements(8,10,11) that influence RSC functionality(12). The ATPase and arm modules sandwich the nucleosome disc with the Snf2 ATP-coupling (SnAC) domain and the finger helix, respectively. The translocase motor of the ATPase module engages with the edge of the nucleosome at superhelical location +2. The mobile ARP module may modulate translocase-nucleosome interactions to regulate RSC activity(5). The RSC-nucleosome structure provides a basis for understanding NDR formation and the structure and function of human SWI/SNF complexes that are frequently mutated in cancer(13).

[more...]

Discovery of a molecular glue promoting CDK12-DDB1 interaction to trigger cyclin K degradation

Elife. 2020. Lv, L et al. Natl Inst Biol Sci, Beijing, Peoples R China.

ABSTRACT: Molecular-glue degraders mediate interactions between target proteins and components of the ubiquitin-proteasome system to cause selective protein degradation. Here, we report a new molecular glue HQ461 discovered by high-throughput screening. Using loss-of-function and gain-of-function genetic screening in human cancer cells followed by biochemical reconstitution, we show that HQ461 acts by promoting an interaction between CDK12 and DDB1-CUL4-RBX1 E3 ubiquitin ligase, leading to polyubiquitination and degradation of CDK12-interacting protein Cyclin K (CCNK).

Degradation of CCNK mediated by HQ461 compromised CDK12 function, leading to reduced phosphorylation of a CDK12 substrate, downregulation of DNA damage response genes, and cell death. Structure-activity relationship analysis of HQ461 revealed the importance of a 5-methylthiazol-2-amine pharmacophore and resulted in an HQ461 derivate with improved potency. Our studies reveal a new molecular glue that recruits its target protein directly to DDB1 to bypass the requirement of a substrate-specific receptor, presenting a new strategy for targeted protein degradation.

[more...]

Molecular architecture of the human 17S U2 snRNP

Nature. 2020. Zhang, ZW et al. MPI Biophys Chem, Dept Struct Dynam, Gottingen, Germany.

ABSTRACT: The U2 small nuclear ribonucleoprotein (snRNP) has an essential role in the selection of the precursor mRNA branch-site adenosine, the nucleophile for the first step of splicing'. Stable addition of U2 during early spliceosome formation requiresthe DEAD-box ATPase PRP5(2-7). Yeast U2 small nuclear RNA (snRNA) nucleotides that form base pairs with the branch site are initially sequestered in a branchpoint-interacting stem-loop (BSL)(8), but whether the human U2 snRNA folds in a similar manner is unknown.

The U2 SF3B1 protein, a common mutational target in haematopoietic cancers(9), contains a HEAT domain (SF3B1(HEAT)) with an open conformation in isolated SF3b(10), but a closed conformation in spliceosomes(11), which is required for stable interaction between U2 and the branch site. Here we report a 3D cryo-electron microscopy structure ofthe human 17S U2 snRNP at a core resolution of 4.1 angstrom and combine it with protein crosslinking data to determine the molecular architecture of this snRNP. Our structure reveals that SF3B1(HEAT) interacts with PRP5 and TAT-SF1, and maintains its open conformation in U2 snRNP, and that U2 snRNA forms a BSL that is sandwiched between PRP5, TAT-SF1 and SF3B1(HEAT). Thus, substantial remodelling of the BSL and displacement of BSL-interacting proteins must occur to allow formation of the U2-branch-site helix. Our studies provide a structural explanation of why TAT-SF1 must be displaced before the stable addition of U2 to the spliceosome, and identify RNP rearrangements facilitated by PRP5 that are required for stable interaction between U2 and the branch site.

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Epitope and Paratope Mapping of PD-1/Nivolumab by Mass Spectrometry-Based Hydrogen--Deuterium Exchange, Cross-linking, and Molecular Docking

Analytical Chemistry. 2020. Zhang, MM et al. Bristol Myers Squibb Co, Nonclin Res & Dev, Pharmaceut Candidate Optimizat, Princeton, NJ 08540 USA.

ABSTRACT: Programmed cell death-1 (PD-1), an antigen co-receptor on cell surfaces, is one of the conspicuous immune checkpoints. Nivolumab, a monoclonal antibody therapeutic approved by the FDA, binds to PD-1 and efficiently blocks its pathways. In this study, an integrated approach was developed to map the epitope/paratope of PD-1/nivolumab. The approach includes hydrogen -deuterium exchange mass spectrometry (HDX-MS) followed by electron-transfer dissociation (ETD), chemical cross-linking, and molecular docking.

HDX-ETD offers some binding-site characterization with amino acid resolution. Chemical cross-linking provides complementary information on one additional epitope (i.e., the BC-loop) and a potential paratope at the N-terminus of the heavy chain. Furthermore, cross-linking identifies another loop region (i.e., the CD-loop) that undergoes a remote conformational change. The distance restraints derived from the cross-links enable building high-confidence models of PD-1/nivolumab, evaluated with respect to a resolved crystal structure. This integrated strategy is an opportunity to characterize comprehensively other antigen-antibody interactions, to enable the understanding of binding mechanisms, and to design future antibody therapeutics.

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Identification of sulfenylated cysteines in Arabidopsis thaliana proteins using a disulfide-linked peptide reporter

Frontiers in plant science. 2020. Wei, B et al. Univ Ghent, Dept Plant Biotechnol & Bioinformat, Ghent, Belgium.

ABSTRACT: In proteins, hydrogen peroxide (H2O2) reacts with redox-sensitive cysteines to form cysteine sulfenic acid, also known asS-sulfenylation. These cysteine oxidation events can steer diverse cellular processes by altering protein interactions, trafficking, conformation, and function. Previously, we had identifiedS-sulfenylated proteins by using a tagged proteinaceous probe based on the yeast AP-1-like (Yap1) transcription factor that specifically reacts with sulfenic acids and traps them through a mixed disulfide bond.

However, the identity of theS-sulfenylated amino acid residues within a protein remained enigmatic. By using the same transgenic YAP1C probe, we present here a technological advancement to identifyin situsulfenylated cysteine sites inArabidopsis thalianacells under control condition and oxidative stress. Briefly, the total extract of transgenic YAP1CA. thalianacells was initially purified on IgG-Sepharose beads, followed by a tryptic digest. Then, the mixed disulfide-linked peptides were further enriched at the peptide level on an anti-YAP1C-derived peptide (C598SEIWDR) antibody. Subsequent mass spectrometry analysis with pLink 2 identified 1,745 YAP1C cross-linked peptides, indicating sulfenylated cysteines in over 1,000 proteins. Approximately 55% of these YAP1C-linked cysteines had previously been reported as redox-sensitive cysteines (S-sulfenylation,S-nitrosylation, and reversibly oxidized cysteines). The presented methodology provides a noninvasive approach to identify sulfenylated cysteines in any species that can be genetically modified.

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Expanding the Depth and Sensitivity of Cross-Link Identification by Differential Ion Mobility Using High-Field Asymmetric Waveform Ion Mobility Spectrometry

Analytical Chemistry. 2020. Schnirch, L et al. Leibniz Forschungsinst Mol Pharmakol FMP, Dept Chem Biol, D-13125 Berlin, Germany.

ABSTRACT: In cross-linking mass spectrometry (XL-MS), the depth and sensitivity of cross-link detection is often limited by the low abundance of cross-links compared to non-cross-linked peptides in the digestion mixture. To improve the identification efficiency of cross-links, here, we present a gas-phase separation strategy using high-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to the Orbitrap Tribrid mass spectrometers. By enabling an additional peptide separation step in the gas phase using the FAIMS device, we increase the number of cross-link identifications by 22% for a medium complex sample and 59% for strong cation exchange-fractionated HEK293T cell lysate in XL-MS experiments using disuccinimidyl sulfoxide (DSSO) cross-linker.

When disuccinimidyl suberate (DSS) cross-linker is in use, we are able to boost cross-link identification by 89% for the medium and 100% for the high complex sample compared to the analyses without FAIMS. Furthermore, we show that, for medium complex samples, FAIMS enables the collection of single-shot XL-MS data with a comparable depth to the corresponding sample fractionated by chromatography-based approaches. Altogether, we demonstrate FAIMS is highly beneficial for XL-MS studies by expanding the proteome coverage of cross-links while improving the efficiency and confidence of cross-link identification.

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Vaccinia Virus Immunomodulator A46: Destructive Interactions with MAL and MyD88 Shown by Negative-Stain Electron Microscopy

Structure. 2020. Azar, DF et al. Med Univ Vienna, Vienna Bioctr, Max Perutz Labs, Dr Bohr Gasse 9-3, A-1030 Vienna, Austria.

ABSTRACT: Vaccinia virus A46 is an anti-inflammatory and non-anti-apoptotic, two-domain member of the poxviral Bcl-2-like protein family that inhibits the cellular innate immune response at the level of the Toll/interleukin-1 receptor (TIR) domain-containing TLR adaptor proteins MAL, MyD88, TRAM, and TRIF. The mechanism of interaction of A46 with its targets has remained unclear. The TIR domains of MAL and MyD88 have been shown to signal by forming filamentous assemblies. We show a clear concentration-dependent destruction of both of these assemblies by A46 by means of negative-stain electron microscopy from molar ratios of 1:15 for MAL and 1:30 for MyD88.

Using targeted mutagenesis and protein-protein crosslinking, we show that A46 interacts with MAL and MyD88 through several facets, including residues on helices alpha 1 and alpha 7 and the C-terminal flexible region. We propose a model in which A46 targets the MAL and MyD88 signalosome intra-strand interfaces and gradually destroys their assemblies in a concentration-dependent manner.

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Integrated structural modeling of full-length LRH-1 reveals inter-domain interactions contribute to receptor structure and function

Structure. 2020. Seacrist, CD et al. Vanderbilt Univ, Dept Pharmacol, Nashville, TN 37235 USA.

ABSTRACT: Liver receptor homolog-1 (LRH-1; NR5A2) is a nuclear receptor that regulates a diverse array of biological processes. In contrast to dimeric nuclear receptors, LRH-1 is an obligate monomer and contains a subtype-specific helix at the C terminus of the DNA-binding domain (DBD), termed FTZ-F1. Although detailed structural information is available for individual domains of LRH-1, it is unknown how these domains exist in the intact nuclear receptor Here, we developed an integrated structural model of human full-length LRH-1 using a combination of HDX-MS, XL-MS, Rosetta computational docking, and SAXS, The model predicts the DBD FTZ-F1 helix directly interacts with ligand binding domain helix 2, We confirmed several other predicted inter-domain interactions via structural and functional analyses.

Comparison between the LRH-1/Dax-1 co-crystal structure and the integrated model predicted and confirmed Dax-1 co-repressor to modulate LRH-1 inter-domain dynamics. Together, these data support individual LRH-1 domains interacting to influence receptor structure and function.

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DNA binding reorganizes the intrinsically disordered C-terminal region of PSC in drosophila PRC1

Journal of Molecular Biology. 2020. Kang, JJ et al. Inst Rech Clin Montreal, 110 Ave Pins Ouest, Montreal, PQ H2W 1R7, Canada.

ABSTRACT: Polycomb Group proteins regulate gene expression by modifying chromatin. Polycomb Repressive Complex 1 (PRC1) has two activities: a ubiquitin ligase activity for histone H2A and a chromatin compacting activity. In Drosophila, the Posterior Sex Combs (PSC) subunit of PRC1 is central to both activities. The N-terminal of PSC assembles into PRC1, including partnering with dRING to form the ubiquitin ligase. The intrinsically disordered C-terminal region of PSC compacts chromatin and inhibits chromatin remodeling and transcription in vitro.

Both regions of PSC are essential in vivo. To understand how these two activities may be coordinated in PRC1, we used crosslinking mass spectrometry to analyze the conformations of the C-terminal region of PSC in PRC1 and how they change on binding DNA. Crosslinking identifies interactions between the C-terminal region of PSC and the core of PRC1, including between N and C-terminal regions of PSC. New contacts and overall more compacted PSC C-terminal region conformations are induced by DNA binding. Protein footprinting of accessible lysine residues reveals an extended, bipartite candidate DNA/chromatin binding surface in the C-terminal region of PSC. Our data suggest a model in which DNA (or chromatin) follows a long path on the flexible disordered region of PSC. Intramolecular interactions of PSC detected by crosslinking can bring the high-affinity DNA/chromatin binding region close to the core of PRC1 without disrupting the interface between the ubiquitin ligase and the nucleosome. Our approach may be applicable to understanding the global organization of other large intrinsically disordered regions that bind nucleic acids. (C) 2020 The Author(s). Published by Elsevier Ltd.

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An experimentally generated peptide database increases the sensitivity of XL-MS with complex samples

Journal of proteomics. 2020. Parfentev, I et al. Max Planck Inst Biophys Chem, Res Grp Bioanalyt Mass Spectrometry, Gottingen, Germany.

ABSTRACT: Cross-linking mass spectrometry (XL-MS) is steadily expanding its range of applications from purified protein complexes to more complex samples like organelles and even entire cells. One main challenge using non-cleavable cross-linkers is the so-called n 2 problem: With linearly increasing database size, the search space for the identification of two covalently linked peptides per spectrum increases quadratically. Here, we report an alternative search strategy that focuses on only those peptides, which were demonstrated to cross-link under the applied experimental conditions.

The performance of a parallel XL-MS experiment using a thiol-cleavable cross-linker enabled the identification of peptides that carried a cleaved cross-link moiety after reduction and hence were involved in cross-linking reactions. Based on these identifications, a peptide database was generated and used for the database search of the actual cross-linking experiment with a non-cleavable cross-linker. This peptide-focused approach was tested on protein complexes with a reported structural model and obtained results corresponded well to a conventional database search. An application of the strategy on in vivo cross-linked Bacillus subtilis and Bacillus cereus cells revealed a five- to tenfold reduction in search time and led to significantly more identifications with the latter species than a search against the entire proteome. Significance: Instead of considering all theoretically cross-linkable peptides in a proteome, identification and prefiltering for a subset of cross-link peptide candidates allows for a dramatically decreased search space. Hence, there is less potential for the random accumulation of false positives ultimately leading to a higher sensitivity in the XL-MS experiment. Using the peptide-focused approach, a cross-linking database search can be conducted in a fraction of time while yielding a similar or higher number of identifications, thereby enabling the cross-linking analysis of samples of mammalian proteome complexity.

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Structure of the human sodium leak channel NALCN in complex with FAM155A

Nature communications. 2020. Xie, JF et al. Westlake Univ, Sch Life Sci, Key Lab Struct Biol Zhejiang Prov, Hangzhou 310024, Zhejiang, Peoples R China.

ABSTRACT: NALCN, a sodium leak channel expressed mainly in the central nervous system, is responsible for the resting Na+ permeability that controls neuronal excitability. Dysfunctions of the NALCN channelosome, NALCN with several auxiliary subunits, are associated with a variety of human diseases. Here, we report the cryo-EM structure of human NALCN in complex with FAM155A at an overall resolution of 3.1 angstroms. FAM155A forms extensive interactions with the extracellular loops of NALCN that may help stabilize NALCN in the membrane.

A Na+ ion-binding site, reminiscent of a Ca2+ binding site in Ca-v channels, is identified in the unique EEKE selectivity filter. Despite its 'leaky' nature, the channel is closed and the intracellular gate is sealed by S6(I), II-III linker and III-IV linker. Our study establishes the molecular basis of Na+ permeation and voltage sensitivity, and provides important clues to the mechanistic understanding of NALCN regulation and NALCN channelosome-related diseases. NALCN, a sodium leak channel, plays a key role in regulating the resting membrane potential and controlling neuronal excitability. Here the authors report a cryo-EM structure of human NALCN in complex with FAM155A, that with complementary functional analyses provide insights on its ion selectivity, voltage sensing and specific interactions with auxiliary subunits.

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Structural insights into the roles of metazoan-specific splicing factors in the human step 1 spliceosome

Molecular Cell. 2020. Bertram, K et al. MPI Biophys Chem, Dept Struct Dynam, Fassberg 11, D-37077 Gottingen, Germany.

ABSTRACT: Human spliceosomes contain numerous proteins absent in yeast, whose functions remain largely unknown. Here we report a 3D cryo-EM structure of the human spliceosomal C complex at 3.4 angstrom core resolution and 4.5-5.7 angstrom at its periphery, and aided by protein crosslinking we determine its molecular architecture. Our structure provides additional insights into the spliceosome's architecture between the catalytic steps of splicing, and how proteins aid formation of the spliceosome's catalytically active RNP (ribonucleoprotein) conformation.

It reveals the spatial organization of the metazoan-specific proteins PPWD1, WDR70, FRG1, and CIR1 in human C complexes, indicating they stabilize functionally important protein domains and RNA structures rearranged/repositioned during the B-act to C transition. Structural comparisons with human B-act, C*, and P complexes reveal an intricate cascade of RNP rearrangements during splicing catalysis, with intermediate RNP conformations not found in yeast, and additionally elucidate the structural basis for the sequential recruitment of metazoan-specific spliceosomal proteins.

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Decision tree searching strategy to boost the identification of cross-linked peptides

Analytical Chemistry. 2020. Huang, R et al. ShanghaiTech Univ, Shanghai Inst Adv Immunochem Studies, Shanghai 201210, Peoples R China.

ABSTRACT: We describe an efficient decision tree searching strategy (DTSS) to boost the identification of cross-linked peptides. The DTSS approach allows the identification of a wealth of complementary information to facilitate the construction of more protein-protein interaction networks for human cell lysate, which was tested by the use of a recently reported cross-linking data set (ACS Cent. Sci. 2019, 5, 1514-1522). A variant of the PhoX-linker, named pDSPE, was synthesized and applied to cross-link Escherichia coli cell lysate to demonstrate that the acquisition of doubly charged ions can significantly improve identification results.

The method can be seamlessly integrated to other search engines to maximize the number of identified cross-links.

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Use of Multiple Ion Fragmentation Methods to Identify Protein Cross-Links and Facilitate Comparison of Data Interpretation Algorithms

Journal of Proteome Research. 2020. Zhao, BQ et al. Indiana Univ, Dept Chem, Bloomington, IN 47405 USA.

ABSTRACT: Multiple ion fragmentation methods involving collision-induced dissociation (CID), higher-energy collisional dissociation (HCD) with regular and very high energy settings, and electron-transfer dissociation with supplementary HCD (EThcD) are implemented to improve the confidence of cross-link identifications. Three different S. cerevisiae proteasome samples cross-linked by diethyl suberthioimidate (DEST) or bis(sulfosuccinimidyl)suberate (BS3) are analyzed. Two approaches are introduced to combine interpretations from the above four methods.

Working with cleavable crosslinkers such as DEST, the first approach searches for cross-link diagnostic ions and consistency among the best interpretations derived from all four MS2 spectra associated with each precursor ion. Better agreement leads to a more definitive identification. Compatible with both cleavable and noncleavable cross-linkers such as BS3, the second approach multiplies scoring metrics from a number of fragmentation experiments to derive an overall best match. This significantly increases the scoring gap between the target and decoy matches. The validity of cross-links fragmented by HCD alone and identified by Kojak, MeroX, pLink, and Xi was evaluated using multiple fragmentation data. Possible ways to improve the identification credibility are discussed. Data are available via ProteomeXchange with identifier PXD018310.

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FGF23 contains two distinct high-affinity binding sites enabling bivalent interactions with $\alpha$-Klotho

PNAS. 2020. Suzuki, Y et al. Yale Univ, Dept Pharmacol, Sch Med, New Haven, CT 06510 USA.

ABSTRACT: The three members of the endocrine-fibroblast growth factor (FGF) family, FGF19, 21, and 23 are circulating hormones that regulate critical metabolic processes. FGF23 stimulates the assembly of a signaling complex composed of alpha-Klotho (KLA) and FGF receptor (FGFR) resulting in kinase activation, regulation of phosphate homeostasis, and vitamin D levels. Here we report that the C-terminal tail of FGF23, a region responsible for KLA binding, contains two tandem repeats, repeat 1 (R1) and repeat 2 (R2) that function as two distinct ligands for KLA.

FGF23 variants with a single KLA binding site, FGF23-R1, FGF23-R2, or FGF23-wild type (WT) with both R1 and R2, bind to KLA with similar binding affinity and stimulate FGFR1 activation and MAPK response. R2 is flanked by two cysteines that form a disulfide bridge in FGF23-WT; disulfide bridge formation in FGF23-WT is dispensable for KLA binding and for cell signaling via FGFRs. We show that FGF23-WT stimulates dimerization and activation of a chimeric receptor molecule composed of the extracellular domain of KLA fused to the cytoplasmic domain of FGFR and employ total internal reflection fluorescence microscopy to visualize individual KLA molecules on the cell surface. These experiments demonstrate that FGF23-WT can act as a bivalent ligand of KLA in the cell membrane. Finally, an engineered Fc-R2 protein acts as an FGF23 antagonist offering new pharmacological intervention for treating diseases caused by excessive FGF23 abundance or activity.

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OpenPepXL: An open-source tool for sensitive identification of cross-linked peptides in XL-MS

Molecular & Cellular Proteomics. 2020. Netz, E et al. Max Planck Inst Dev Biol, Biomol Interact, Tubingen, Germany.

ABSTRACT: XL-MS has been recognized as an effective source of information about protein structures and interactions. OpenPepXL is a sensitive XL-MS identification software that reports from 7% to 40% more structurally validated cross-links than other tools on data sets with available high-resolution structures for cross-link validation. It is open source and has been built as part of the OpenMS suite of tools. OpenPepXL supports all common operating systems and open data formats. Cross-linking MS (XL-MS) has been recognized as an effective source of information about protein structures and interactions.

In contrast to regular peptide identification, XL-MS has to deal with a quadratic search space, where peptides from every protein could potentially be cross-linked to any other protein. To cope with this search space, most tools apply different heuristics for search space reduction. We introduce a new open-source XL-MS database search algorithm, OpenPepXL, which offers increased sensitivity compared with other tools. OpenPepXL searches the full search space of an XL-MS experiment without using heuristics to reduce it. Because of efficient data structures and built-in parallelization OpenPepXL achieves excellent runtimes and can also be deployed on large compute clusters and cloud services while maintaining a slim memory footprint. We compared OpenPepXL to several other commonly used tools for identification of noncleavable labeled and label-free cross-linkers on a diverse set of XL-MS experiments. In our first comparison, we used a data set from a fraction of a cell lysate with a protein database of 128 targets and 128 decoys. At 5% FDR, OpenPepXL finds from 7% to over 50% more unique residue pairs (URPs) than other tools. On data sets with available high-resolution structures for cross-link validation OpenPepXL reports from 7% to over 40% more structurally validated URPs than other tools. Additionally, we used a synthetic peptide data set that allows objective validation of cross-links without relying on structural information and found that OpenPepXL reports at least 12% more validated URPs than other tools. It has been built as part of the OpenMS suite of tools and supports Windows, macOS, and Linux operating systems. OpenPepXL also supports the MzIdentML 1.2 format for XL-MS identification results. It is freely available under a three-clause BSD license at .

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Mechanism of protein-guided folding of the active site U2/U6 RNA during spliceosome activation

Science (New York, N.Y.). 2020. Townsend, Cole et al. Cellular Biochemistry, MPI for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.

ABSTRACT: Spliceosome activation involves extensive protein and RNA rearrangements that lead to formation of a catalytically active U2/U6 RNA structure. At present, little is known about the assembly pathway of the latter and the mechanism whereby proteins aid its proper folding. Here, we report the cryo-electron microscopy structures of two human, activated spliceosome precursors (that is, pre-Bact complexes) at core resolutions of 3.9 and 4.2 angstroms. These structures elucidate the order of the numerous protein exchanges that occur during activation, the mutually exclusive interactions that ensure the correct order of ribonucleoprotein rearrangements needed to form the U2/U6 catalytic RNA, and the stepwise folding pathway of the latter.

Structural comparisons with mature Bact complexes reveal the molecular mechanism whereby a conformational change in the scaffold protein PRP8 facilitates final three-dimensional folding of the U2/U6 catalytic RNA. Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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Defining the architecture of the human TIM22 complex by chemical crosslinking

FEBS Letters. 2020. Valpadashi, A et al. Univ Med Ctr Gottingen, Dept Cellular Biochem, Humboldtallee 23, D-37073 Gottingen, Germany.

ABSTRACT: The majority of mitochondrial proteins are nuclear encoded and imported into mitochondria as precursor proteins via dedicated translocases. The translocase of the inner membrane 22 (TIM22) is a multisubunit molecular machine specialized for the translocation of hydrophobic, multi-transmembrane-spanning proteins with internal targeting signals into the inner mitochondrial membrane. Here, we undertook a crosslinking-mass spectrometry (XL-MS) approach to determine the molecular arrangement of subunits of the human TIM22 complex.

Crosslinking of the isolated TIM22 complex using the BS3 crosslinker resulted in the broad generation of crosslinks across the majority of TIM22 components, including the small TIM chaperone complex. The crosslinking data uncovered several unexpected features, opening new avenues for a deeper investigation into the steps required for TIM22-mediated translocation in humans.

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Dynamic folding modulation generates FGF21 variant against diabetes

EMBO reports. 2020. Zhu, Lei et al. High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy ofSciences, Hefei, China

ABSTRACT: Fibroblast growth factor 21 (FGF21) is a regulator of glucose and lipid metabolism. It has been widely considered as a promising candidate for the treatment of type 2 diabetes mellitus (T2DM) and other related metabolic disorders. However, lack of structural and dynamic information has limited FGF21-based drug development. Here, using nuclear magnetic resonance (NMR) spectroscopy, we determine the structure of FGF21 and find that its non-canonical flexible beta-trefoil conformation affects the folding of beta2-beta3 hairpin and further overall protein stability.

To modulate folding dynamics, we designed an FGF21-FGF19 chimera, FGF21SS . As expected, FGF21SS shows better thermostability without inducing hepatocyte proliferation. Functional characterization of FGF21SS shows its better insulin sensitivity, reduced inflammation in 3T3-L1 adipocytes, and lower blood glucose and insulin levels in ob/ob mice compared with wild type. Our dynamics-based rational design provides a promising approach for FGF21-based therapeutic development against T2DM. © 2020 The Authors.

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Dimerization regulates the human APC/C-associated ubiquitin-conjugating enzyme UBE2S

Science Signaling. 2020. Liess, AKL et al. Univ Wurzburg, Rudolf Virchow Ctr Integrat & Translat Bioimaging, D-97080 Wurzburg, Germany.

ABSTRACT: At the heart of protein ubiquitination cascades, ubiquitin-conjugating enzymes (E2s) form reactive ubiquitin-thioester intermediates to enable efficient transfer of ubiquitin to cellular substrates. The precise regulation of E2s is thus crucial for cellular homeostasis, and their deregulation is frequently associated with tumorigenesis. In addition to driving substrate ubiquitination together with ubiquitin ligases (E3s), many E2s can also autoubiquitinate, thereby promoting their own proteasomal turnover.

To investigate the mechanisms that balance these disparate activities, we dissected the regulatory dynamics of UBE2S, a human APC/C-associated E2 that ensures the faithful ubiquitination of cell cycle regulators during mitosis. We uncovered a dimeric state of UBE2S that confers auto-inhibition by blocking a catalytically critical ubiquitin binding site. Dimerization is stimulated by the lysine-rich carboxyl-terminal extension of UBE2S that is also required for the recruitment of this E2 to the APC/C and is autoubiquitinated as substrate abundance becomes limiting. Consistent with this mechanism, we found that dimerization-deficient UBE2S turned over more rapidly in cells and did not promote mitotic slippage during prolonged drug-induced mitotic arrest. We propose that dimerization attenuates the autoubiquitination-induced turnover of UBE2S when the APC/C is not fully active. More broadly, our data illustrate how the use of mutually exclusive macromolecular interfaces enables modulation of both the activities and the abundance of E2s in cells to facilitate precise ubiquitin signaling.

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Switch from Polymorphic to Homogenous Self-Assembly of Virus-Like Particles of Simian Virus 40 through Double-Cysteine Substitution

Small. 2020. Xu, CC et al. Guangzhou Med Univ, Guangzhou Women & Childrens Med Ctr, Guangzhou 510623, Peoples R China.

ABSTRACT: Self-assembled virus-like particles (VLPs) hold great potential as natural nanomaterials for applications in many fields. For such purposes, monodisperse size distribution is a desirable property. However, the VLPs of simian virus 40 (SV40), a representative VLP platform, are characterized by polymorphism. In an attempt to eliminate the polymorphism, 15 mutants of the VLP subunit (VP1) are constructed through the substitution of double cysteines at the VP1 pentamer interfaces, generating a group of VLPs with altered size distributions.

One of the mutants, SS2 (L102C/P300C), specifically forms homogenousT = 1-like tiny VLPs of 24 +/- 3 nm in diameter. Moreover, the stability of the SS2 VLPs is markedly enhanced compared with that of wild-type VLPs. The homogeneous self-assembly and stability enhancement of SS2 VLPs can be attributed to the new disulfide bonds contributed by Cys102 and Cys300, which are identified by mass spectrometry and explored by molecular dynamics simulations. Endocytosis inhibition assays indicate that SS2 VLPs, like the polymorphic wild-type VLPs, preserve the multipathway feature of cellular uptake. SS2 VLPs may serve as an evolved version of SV40 VLPs in future studies and applications. The findings of this work would be useful for the design and fabrication of VLP-based materials and devices.

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Mapping the native interaction surfaces of PREP1 with PBX1 by cross-linking mass-spectrometry and mutagenesis

Scientific Reports. 2020. Bruckmann, C et al. IFOM Fdn FIRC, Inst Mol Oncol, Via Adamello 16, I-20139 Milan, Italy.

ABSTRACT: Both onco-suppressor PREP1 and the oncogene MEIS1 bind to PBX1. This interaction stabilizes the two proteins and allows their translocation into the nucleus and thus their transcriptional activity. Here, we have combined cross-linking mass-spectrometry and systematic mutagenesis to detail the binding geometry of the PBX1-PREP1 (and PBX1-MEIS1) complexes, under native in vivo conditions. The data confirm the existence of two distinct interaction sites within the PBC domain of PBX1 and unravel differences among the highly similar binding sites of MEIS1 and PREP1.

The HR2 domain has a fundamental role in binding the PBC-B domain of PBX1 in both PREP1 and MEIS1. The HR1 domain of MEIS1, however, seem to play a less stringent role in PBX1 interaction with respect to that of PREP1. This difference is also reflected by the different binding affinity of the two proteins to PBX1. Although partial, this analysis provides for the first time some ideas on the tertiary structure of the complexes not available before. Moreover, the extensive mutagenic analysis of PREP1 identifies the role of individual hydrophobic HR1 and HR2 residues, both in vitro and in vivo.

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Glucocorticoid receptor complexes form cooperatively with the Hsp90 co-chaperones Pp5 and FKBPs

Scientific Reports. 2020. Kaziales, A et al. Tech Univ Munich, Dept Chem, Ctr Integrated Prot Sci Munich, Lichtenbergstr 4, D-85748 Garching, Germany.

ABSTRACT: The function of steroid receptors in the cell depends on the chaperone machinery of Hsp90, as Hsp90 primes steroid receptors for hormone binding and transcriptional activation. Several conserved proteins are known to additionally participate in receptor chaperone assemblies, but the regulation of the process is not understood in detail. Also, it is unknown to what extent the contribution of these cofactors is conserved in other eukaryotes. We here examine the reconstituted C. elegans and human chaperone assemblies.

We find that the nematode phosphatase PPH-5 and the prolyl isomerase FKB-6 facilitate the formation of glucocorticoid receptor (GR) complexes with Hsp90. Within these complexes, Hsp90 can perform its closing reaction more efficiently. By combining chemical crosslinking and mass spectrometry, we define contact sites within these assemblies. Compared to the nematode Hsp90 system, the human system shows less cooperative client interaction and a stricter requirement for the co-chaperone p23 to complete the closing reaction of GRHsp90Pp5/Fkbp51/Fkbp52 complexes. In both systems, hormone binding to GR is accelerated by Hsp90 alone and in the presence of its cofactors. Our results show that cooperative complex formation and hormone binding patterns are, in many aspects, conserved between the nematode and human systems.

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Structural basis of human full-length kindlin-3 homotrimer in an auto-inhibited state

PLOS Biology. 2020. Bu, WT et al. Nanyang Technol Univ, Sch Biol Sci, Singapore, Singapore.

ABSTRACT: Kindlin-1, -2, and -3 directly bind integrin beta cytoplasmic tails to regulate integrin activation and signaling. Despite their functional significance and links to several diseases, structural information on full-length kindlin proteins remains unknown. Here, we report the crystal structure of human full-length kindlin-3, which reveals a novel homotrimer state. Unlike kindlin-3 monomer, which is the major population in insect and mammalian cell expression systems, kindlin-3 trimer does not bind integrin beta cytoplasmic tail as the integrin-binding pocket in the F3 subdomain of 1 protomer is occluded by the pleckstrin homology (PH) domain of another protomer, suggesting that kindlin-3 is auto-inhibited upon trimer formation.

This is also supported by functional assays in which kindlin-3 knockout K562 erythroleukemia cells reconstituted with the mutant kindlin-3 containing trimer-disrupting mutations exhibited an increase in integrin-mediated adhesion and spreading on fibronectin compared with those reconstituted with wild-type kindlin-3. Taken together, our findings reveal a novel mechanism of kindlin auto-inhibition that involves its homotrimer formation.

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Generation of aggregates of $\alpha$-lactalbumin by UV-B light exposure

Journal of Agricultural and Food Chemistry. 2020. Zhao, ZC et al. Univ Copenhagen, Fac Sci, Dept Food Sci, DK-1958 Frederiksberg C, Denmark.

ABSTRACT: Whey proteins are widely used as ingredients in the form of aggregates to obtain certain functionalities in food applications. The aim of this study was to understand how UV illumination generates aggregates of alpha-lactalbumin (alpha-LA) as an alternative to heat treatments traditionally used for industrial production of protein aggregates. Absorption of UV light by alpha-LA caused cleavage of disulfide bonds and release of thiol groups, which resulted in primarily disulfide-mediated aggregation.

This process mediated efficient aggregation with up to 98% monomer conversion into aggregates through formation of intermolecular disulfide bonds, while only minor levels of nonreducible cross-links were observed. SDS-PAGE analysis revealed that illumination led to formation of dimeric, trimeric, and oligomeric forms of alpha-LA. LC-MS/MS analysis showed that all of the four native disulfide bonds in alpha-LA were cleaved by UV illumination but to different extents, and the extent of cleavage was found to be higher in the absence of calcium. Seventeen different non-native disulfides were formed after 24 h of UV illumination. Two dityrosine bonds were identified (Tyr103-Tyr103 and Tyr36-Tyr103) alongside ditryptophan (Trp118-Trp118) and tyrosine-tryptophan (Tyr50-Trp60) cross-links. In addition, Trp60, Trp118, Cys73, Cys91, Cys120, Phe80, Met90, His68, and His107 were found to be oxidized up to 12% as compared to a nonilluminated control. Our work illustrates that light exposure can be used for generation of alpha-LA aggregates, but optimization of the illumination conditions is required to reduce oxidative damage to Trp, Cys, Phe, Met, and His residues.

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Structure of complete Pol II--DSIF--PAF--SPT6 transcription complex reveals RTF1 allosteric activation

Nature Structural & Molecular Biology. 2020. Vos, SM et al. Max Planck Inst Biophys Chem, Dept Mol Biol, Gottingen, Germany.

ABSTRACT: Transcription by RNA polymerase II (Pol II) is carried out by an elongation complex. We previously reported an activated porcine Pol II elongation complex, EC*, encompassing the human elongation factors DSIF, PAF1 complex (PAF) and SPT6. Here we report the cryo-EM structure of the complete EC* that contains RTF1, a dissociable PAF subunit critical for chromatin transcription. The RTF1 Plus3 domain associates with Pol II subunit RPB12 and the phosphorylated C-terminal region of DSIF subunit SPT5. RTF1 also forms four alpha-helices that extend from the Plus3 domain along the Pol II protrusion and RPB10 to the polymerase funnel.

The C-terminal 'fastener' helix retains PAF and is followed by a 'latch' that reaches the end of the bridge helix, a flexible element of the Pol II active site. RTF1 strongly stimulates Pol II elongation, and this requires the latch, possibly suggesting that RTF1 activates transcription allosterically by influencing Pol II translocation. Cryo-EM elucidation of a fully reconstituted Pol II-DSF-PAF1-SPT6 elongation complex defines the position of PAF1 subunit RTF1 and reveals contacts with the Pol II bridge helix that may allosterically stimulate transcription elongation.

[more...]

Functional and mass spectrometric evaluation of an anti-tick antigen based on the P0 peptide conjugated to Bm86 protein

Pathogens. 2020. Mallon, AR et al. Ctr Genet Engn & Biotechnol CIGB, Anim Biotechnol Dept, Havana 10600, Cuba.

ABSTRACT: A synthetic 20 amino acid peptide of the ribosomal protein P0 from ticks, when conjugated to keyhole limpet hemocyanin fromMegathura crenulataand used as an immunogen againstRhipicephalus microplusandRhipicephalus sanguineuss.l. species, has shown efficacies of around 90%. There is also experimental evidence of a high efficacy of this conjugate againstAmblyomma mixtumandIxodes ricinusspecies, which suggest that this antigen could be a good broad-spectrum anti-tick vaccine candidate. In this study, the P0 peptide (pP0) was chemically conjugated to Bm86 as a carrier protein.

SDS-PAGE analysis of this conjugate demonstrated that it is highly heterogeneous in size, carrying from 1 to 18 molecules of pP0 per molecule of Bm86. Forty-nine out of the 54 lysine residues and the N-terminal end of Bm86 were found partially linked to pP0 by using LC-MS/MS analysis and the combination of four different softwares. Several post-translational modifications of Bm86 protein were also identified by mass spectrometry. High immunogenicity and efficacy were achieved when dogs and cattle were vaccinated with the pP0-Bm86 conjugate and challenged withR. sanguineuss.l. andR. microplus, respectively. These results encourage the development of this antigen with promising possibilities as an anti-tick vaccine.

[more...]

A cross-linking mass spectrometry approach defines protein interactions in yeast mitochondria

Molecular & Cellular Proteomics. 2020. Linden, A et al. Max Planck Inst Biophys Chem, Bioanalyt Mass Spectrometry Grp, Gottingen, Germany.

ABSTRACT: Protein cross-linking and the analysis of cross-linked peptides by mass spectrometry is currently receiving much attention. Not only is this approach applied to isolated complexes to provide information about spatial arrangements of proteins, but it is also increasingly applied to entire cells and their organelles. As in quantitative proteomics, the application of isotopic labeling further makes it possible to monitor quantitative changes in the protein-protein interactions between different states of a system.

Here, we cross-linked mitochondria from Saccharomyces cerevisiae grown on either glycerol- or glucose-containing medium to monitor protein-protein interactions under non-fermentative and fermentative conditions. We investigated qualitatively the protein-protein interactions of the 400 most abundant proteins applying stringent data-filtering criteria, i.e. a minimum of two cross-linked peptide spectrum matches and a cut-off in the spectrum scoring of the used search engine. The cross-linker BS3 proved to be equally suited for connecting proteins in all compartments of mitochondria when compared with its waterinsoluble but membrane-permeable derivative DSS. We also applied quantitative cross-linking to mitochondria of both the growth conditions using stable-isotope labeled BS3. Significant differences of cross-linked proteins under glycerol and glucose conditions were detected, however, mainly because of the different copy numbers of these proteins in mitochondria under both the conditions. Results obtained from the glycerol condition indicate that the internal NADH:ubiquinone oxidoreductase Ndi1 is part of an electron transport chain supercomplex. We have also detected several hitherto uncharacterized proteins and identified their interaction partners. Among those, Min8 was found to be associated with cytochrome c oxidase. BN-PAGE analyses of min8 Delta mitochondria suggest that Min8 promotes the incorporation of Cox12 into cytochrome c oxidase.

[more...]

Structure of the transcription coactivator SAGA

Nature. 2020. Haibo Wang et al. Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Göttingen, Germany

ABSTRACT: Gene transcription by RNA polymerase II is regulated by activator proteins that recruit the coactivator complexes SAGA (Spt-Ada-Gcn5-acetyltransferase)(1,2) and transcription factor IID (TFIID)(2-4). SAGA is required for all regulated transcription(5) and is conserved among eukaryotes(6). SAGA contains four modules(7-9): the activator-binding Tra1 module, the core module, the histone acetyltransferase (HAT) module and the histone deubiquitination (DUB) module. Previous studies provided partial structures(10-14), but the structure of the central core module is unknown.

Here we present the cryo-electron microscopy structure of SAGA from the yeast Saccharomyces cerevisiae and resolve the core module at 3.3 angstrom resolution. The core module consists of subunits Taf5, Sgf73 and Spt20, and a histone octamer-like fold. The octamer-like fold comprises the heterodimers Taf6-Taf9, Taf10-Spt7 and Taf12-Ada1, and two histone-fold domains in Spt3. Spt3 and the adjacent subunit Spt8 interact with the TATA box-binding protein (TBP)(2,7,15-17). The octamer-like fold and its TBP-interacting region are similar in TFIID, whereas Taf5 and the Taf6 HEAT domain adopt distinct conformations. Taf12 and Spt20 form flexible connections to the Tra1 module, whereas Sgf73 tethers the DUB module. Binding of a nucleosome to SAGA displaces the HAT and DUB modules from the core-module surface, allowing the DUB module to bind one face of an ubiquitinated nucleosome.

[more...]

A synthetic peptide library for benchmarking crosslinking-mass spectrometry search engines for proteins and protein complexes

Nature Communications. 2020. Beveridge, R et al. Vienna Bioctr VBC, Res Inst Mol Pathol IMP, Campus Vienna Bioctr 1, A-1030 Vienna, Austria.

ABSTRACT: Crosslinking-mass spectrometry (XL-MS) serves to identify interaction sites between proteins. Numerous search engines for crosslink identification exist, but lack of ground truth samples containing known crosslinks has precluded their systematic validation. Here we report on XL-MS data arising from measuring synthetic peptide libraries that provide the unique benefit of knowing which identified crosslinks are true and which are false. The data are analysed with the most frequently used search engines and the results filtered to an estimated false discovery rate of 5%.

We find that the actual false crosslink identification rates range from 2.4 to 32%, depending on the analysis strategy employed. Furthermore, the use of MS-cleavable crosslinkers does not reduce the false discovery rate compared to non-cleavable crosslinkers. We anticipate that the datasets acquired during this research will further drive optimisation and development of XL-MS search engines, thereby advancing our understanding of vital biological interactions.

[more...]

The acetyltransferase Eco1 elicits cohesin dimerization during S phase

Journal of Biological Chemistry. 2020. Shi, D et al. China Agr Univ, State Key Lab Agrobiotechnol, Beijing 100193, Peoples R China.

ABSTRACT: Cohesin is a DNA-associated protein complex that forms a tripartite ring controlling sister chromatid cohesion, chromosome segregation and organization, DNA replication, and gene expression. Sister chromatid cohesion is established by the protein acetyltransferase Eco1, which acetylates two conserved lysine residues on the cohesin subunit Smc3 and thereby ensures correct chromatid separation in yeast (Saccharomyces cerevisiae) and other eukaryotes. However, the consequence of Eco1-catalyzed cohesin acetylation is unknown, and the exact nature of the cohesive state of chromatids remains controversial.

Here, we show that self-interactions of the cohesin subunits Scc1/Rad21 and Scc3 occur in a DNA replication?coupled manner in both yeast and human cells. Using cross-linking MS-based and in vivo disulfide cross-linking analyses of purified cohesin, we show that a subpopulation of cohesin may exist as dimers. Importantly, upon temperature-sensitive and auxin-induced degron-mediated Eco1 depletion, the cohesin-cohesin interactions became significantly compromised, whereas deleting either the deacetylase Hos1 or the Eco1 antagonist Wpl1/Rad61 increased cohesin dimer levels by ?20%. These results indicate that cohesin dimerizes in the S phase and monomerizes in mitosis, processes that are controlled by Eco1, Wpl1, and Hos1 in the sister chromatid cohesion-dissolution cycle. These findings suggest that cohesin dimerization is controlled by the cohesion cycle and support the notion that a double-ring cohesin model operates in sister chromatid cohesion.

[more...]

Rett syndrome-causing mutations compromise MeCP2-mediated liquid--liquid phase separation of chromatin

Cell Research. 2020. Wang, L et al. Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Tsinghua Univ Peking Univ Joint Ctr Life Sci, Sch Life Sci,Beijing Frontier Res Ctr Biol Struct, Beijing, Peoples R China.

ABSTRACT: Rett syndrome (RTT), a severe postnatal neurodevelopmental disorder, is caused by mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). MeCP2 is a chromatin organizer regulating gene expression. RTT-causing mutations have been shown to affect this function. However, the mechanism by which MeCP2 organizes chromatin is unclear. In this study, we found that MeCP2 can induce compaction and liquid-liquid phase separation of nucleosomal arrays in vitro, and DNA methylation further enhances formation of chromatin condensates by MeCP2.

Interestingly, RTT-causing mutations compromise MeCP2-mediated chromatin phase separation, while benign variants have little effect on this process. Moreover, MeCP2 competes with linker histone H1 to form mutually exclusive chromatin condensates in vitro and distinct heterochromatin foci in vivo. RTT-causing mutations reduce or even abolish the ability of MeCP2 to compete with histone H1 and to form chromatin condensates. Together, our results identify a novel mechanism by which phase separation underlies MeCP2-mediated heterochromatin formation and reveal the potential link between this process and the pathology of RTT.

[more...]

Discovery of a regulatory subunit of the yeast fatty acid synthase

Cell. 2020. Singh, K et al. Max Planck Inst Biophys Chem, Dept Struct Dynam, D-37077 Gottingen, Germany.

ABSTRACT: Fatty acid synthases (FASs) are central to metabolism but are also of biotechnological interest for the production of fine chemicals and biofuels from renewable resources. During fatty acid synthesis, the growing fatty acid chain is thought to be shuttled by the dynamic acyl carrier protein domain to several enzyme active sites. Here, we report the discovery of a gamma subunit of the 2.6 megadalton alpha(6)-beta(6) S. cerevisiae FAS, which is shown by high-resolution structures to stabilize a rotated FAS conformation and rearrange ACP domains from equatorial to axial positions.

The gamma subunit spans the length the FAS inner cavity, impeding reductase activities of FAS, regulating NADPH turnover by kinetic hysteresis at the ketoreductase, and suppressing off-pathway reactions at the enoylreductase. The gamma subunit delineates the functional compartment within FAS. As a scaffold, it may be exploited to incorporate natural and designed enzymatic activities that are not present in natural FAS.

[more...]

A receptor for the complement regulator factor H increases transmission of trypanosomes to tsetse flies

Nature Communications. 2020. Macleod, OJS et al. Univ Cambridge, Dept Biochem, Tennis Court Rd, Cambridge CB2 1QW, England.

ABSTRACT: Persistent pathogens have evolved to avoid elimination by the mammalian immune system including mechanisms to evade complement. Infections with African trypanosomes can persist for years and cause human and animal disease throughout sub-Saharan Africa. It is not known how trypanosomes limit the action of the alternative complement pathway. Here we identify an African trypanosome receptor for mammalian factor H, a negative regulator of the alternative pathway. Structural studies show how the receptor binds ligand, leaving inhibitory domains of factor H free to inactivate complement C3b deposited on the trypanosome surface.

Receptor expression is highest in developmental stages transmitted to the tsetse fly vector and those exposed to blood meals in the tsetse gut. Receptor gene deletion reduced tsetse infection, identifying this receptor as a virulence factor for transmission. This demonstrates how a pathogen evolved a molecular mechanism to increase transmission to an insect vector by exploitation of a mammalian complement regulator.

[more...]

Integrative structure and function of the yeast exocyst complex

Protein Science. 2020. Ganesan, SJ et al. Univ Calif San Francisco, Dept Bioengn & Therapeut Sci, Byers Hall,1700 4th St,Suite 503B, San Francisco, CA 94158 USA.

ABSTRACT: Exocyst is an evolutionarily conserved hetero-octameric tethering complex that plays a variety of roles in membrane trafficking, including exocytosis, endocytosis, autophagy, cell polarization, cytokinesis, pathogen invasion, and metastasis. Exocyst serves as a platform for interactions between the Rab, Rho, and Ral small GTPases, SNARE proteins, and Sec1/Munc18 regulators that coordinate spatial and temporal fidelity of membrane fusion. However, its mechanism is poorly described at the molecular level.

Here, we determine the molecular architecture of the yeast exocyst complex by an integrative approach, based on a 3D density map from negative-stain electron microscopy (EM) at similar to 16 angstrom resolution, 434 disuccinimidyl suberate and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride cross-links from chemical-crosslinking mass spectrometry, and partial atomic models of the eight subunits. The integrative structure is validated by a previously determined cryo-EM structure, cross-links, and distances from in vivo fluorescence microscopy. Our subunit configuration is consistent with the cryo-EM structure, except for Sec5. While not observed in the cryo-EM map, the integrative model localizes the N-terminal half of Sec3 near the Sec6 subunit. Limited proteolysis experiments suggest that the conformation of Exo70 is dynamic, which may have functional implications for SNARE and membrane interactions. This study illustrates how integrative modeling based on varied low-resolution structural data can inform biologically relevant hypotheses, even in the absence of high-resolution data.

[more...]

Molecular topology of RNA polymerase I upstream activation factor

Molecular and Cellular Biology. 2020. Knutson, BA et al. SUNY Upstate Med Univ, Dept Biochem & Mol Biol, Syracuse, NY 13210 USA.

ABSTRACT: Upstream activation factor (UAF) is a multifunctional transcription factor in Saccharomyces cerevisiae that plays dual roles in activating RNA polymerase I (Pol I) transcription and repression of Pol II. For Pol I, UAF binds to a specific upstream element in the ribosomal DNA (rDNA) promoter and interacts with two other Pol I initiation factors, the TATA-binding protein (TBP) and core factor (CF). We used an integrated combination of chemical cross-linking mass spectrometry (CXMS), molecular genetics, protein biochemistry, and structural modeling to understand the topological framework responsible for UAF complex formation.

Here, we report the molecular topology of the UAF complex, describe new structural and functional domains that play roles in UAF complex integrity, assembly, and biological function, and provide roles for previously identified UAF domains that include the Rrn5 SANT and histone fold domains. We highlight the role of new domains in Uaf30 that include an N-terminal winged helix domain and a disordered tethering domain as well as a BORCS6-like domain found in Rrn9. Together, our results reveal a unique network of topological features that coalesce around a histone tetramer-like core to form the dual-function UAF complex.

[more...]

Evidence of allosteric coupling between substrate binding and adx recognition in the vitamin D carbon-24 hydroxylase CYP24A1

Biochemistry. 2020. Kumar, A et al. Univ Buffalo, Jacobs Sch Med, Dept Biochem, Buffalo, NY 14203 USA.

ABSTRACT: Metabolic inactivation of 1,25(OH)2D3 requires molecular recognition between the mitochondrial enzyme cytochrome P450 24A1 (CYP24A1) and its cognate redox partner adrenodoxin (Adx). Recent evidence supports a model of CYP24A1 function in which substrate binding and Adx recognition are structurally linked. However, the details of this allosteric connection are not clear. In this study, we utilize chemical cross-linking coupled to mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and CYP24A1 functional assays to inform a working model of a CYP24A1Adx complex.

We report that differential cross-linking internal to CYP24A1 points toward an Adx-induced conformational change that perturbs the F and G helices, which are required for substrate binding. Moreover, the modeled complex suggests that a semiconserved nonpolar interaction at the interface may influence CYP24A1 regioselectivity. Taken together, these findings contribute to our understanding of Adx recognition in a critical vitamin D-inactivating enzyme and provide broader insight regarding the variability inherent in CYPAdx interactions.

[more...]

Fragment mass spectrum prediction facilitates site localization of phosphorylation

Journal of Proteome Research. 2020. Yang, Y et al. Fudan Univ, Dept Chem, Shanghai 200000, Peoples R China.

ABSTRACT: Liquid chromatography tandem mass spectrometry (LCMS/MS) has been the most widely used technology for phosphoproteomics studies. As an alternative to database searching and probability-based phosphorylation site localization approaches, spectral library searching has been proved to be effective in the identification of phosphopeptides. However, incompletion of experimental spectral libraries limits the identification capability. Herein, we utilize MS/MS spectrum prediction coupled with spectral matching for site localization of phosphopeptides.

In silico MS/MS spectra are generated from peptide sequences by deep learning/machine learning models trained with nonphosphopeptides. Then, mass shift according to phosphorylation sites, phosphoric acid neutral loss, and a "budding" strategy are adopted to adjust the in silico mass spectra. In silico MS/MS spectra can also be generated in one step for phosphopeptides using models trained with phosphopeptides. The method is benchmarked on data sets of synthetic phosphopeptides and is used to process real biological samples. It is demonstrated to be a method requiring only computational resources that supplements the probability-based approaches for phosphorylation site localization of singly and multiply phosphorylated peptides.

[more...]

Jasmonate-independent regulation of digestive enzyme activity in the carnivorous butterwort Pinguicula$\times$ Tina

Journal of experimental botany. 2020. Kocab, O et al. Palacky Univ, Fac Sci, Ctr Reg Hana Biotechnol & Agr Res, Dept Biophys, Slechtitelu 27, CZ-78371 Olomouc, Czech Republic

ABSTRACT: Carnivorous plants within the order Caryophyllales use jasmonates, a class of phytohormone, in the regulation of digestive enzyme activities. We used the carnivorous butterwort Pinguicula x Tina from the order Lamiales to investigate whether jasmonate signaling is a universal and ubiquitous signaling pathway that exists outside the order Caryophyllales. We measured the electrical signals, enzyme activities, and phytohormone tissue levels in response to prey capture. Mass spectrometry was used to identify proteins in the digestive secretion.

We identified eight enzymes in the digestive secretion, many of which were previously found in other genera of carnivorous plants. Among them, alpha-amylase is unique in carnivorous plants. Enzymatic activities increased in response to prey capture; however, the tissue content of jasmonic acid and its isoleucine conjugate remained rather low in contrast to the jasmonate response to wounding. Enzyme activities did not increase in response to the exogenous application of jasmonic acid or coronatine. Whereas similar digestive enzymes were co-opted from plant defense mechanisms among carnivorous plants, the mode of their regulation differs. The butterwort has not co-opted jasmonate signaling for the induction of enzyme activities in response to prey capture. Moreover, the presence of alpha-amylase in digestive fluid of P. x Tina, which has not been found in other genera of carnivorous plants, might indicate that non-defense-related genes have also been co-opted for carnivory.

[more...]

Identification and dereplication of endophytic Colletotrichum strains by MALDI TOF mass spectrometry and molecular networking

Scientific reports. 2020. Barthelemy, M et al. Univ Paris Saclay, Inst Chim Subst Nat, CNRS, UPR 2301, Ave Terrasse, F-91198 Gif Sur Yvette, France.

ABSTRACT: The chemical diversity of biologically active fungal strains from 42 Colletotrichum, isolated from leaves of the tropical palm species Astrocaryum sciophilum collected in pristine forests of French Guiana, was investigated. The collection was first classified based on protein fingerprints acquired by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) correlated with cytotoxicity. Liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS) data from ethyl acetate extracts were acquired and processed to generate a massive molecular network (MN) using the MetGem software.

From five Colletotrichum strains producing cytotoxic specialized metabolites, we predicted the occurrence of peptide and cytochalasin analogues in four of them by MN, including a similar ion clusters in the MN algorithm provided by MetGem software. Chemoinformatics predictions were fully confirmed after isolation of three pentacyclopeptides (cyclo(Phe-Leu-Leu-Leu-Val), cyclo(Phe-Leu-Leu-Leu-Leu) and cyclo(Phe-Leu-Leu-Leu-Ile)) and two cytochalasins (cytochalasin C and cytochalasin D) exhibiting cytotoxicity at the micromolar concentration. Finally, the chemical study of the last active cytotoxic strain BSNB-0583 led to the isolation of four colletamides bearing an identical decadienamide chain.

[more...]

Development of a Sample-Preparation Workflow for Sulfopeptide Enrichment: From Target Analysis to Challenges in Shotgun Sulfoproteomics

Analytical Chemistry. 2020. Capriotti, AL et al. Sapienza Univ Roma, Dept Chem, I-00185 Rome, Italy.

ABSTRACT: Protein tyrosine O-sulfation is an important post-translational modification, as it has been correlated to inflammation, virus infection, and signal pathways. Nevertheless, methods for the characterization of protein sulfation by sulfopeptide enrichment are currently limited. In this Article, two standard compounds, representative of mono- and disulfated peptides, were used to compare the enrichment capabilities of five sorbent materials: two commercial weak anion-exchange mixed-mode sorbents (Strata X-AW and Oasis WAX) and three phosphopeptide enrichment materials based on affinity chromatography to either immobilized metals (IMAC) or metal oxides, i.e., Fe3+, TiO2, or Ti4+.

The sulfopeptides were analyzed by ultrahigh-performance liquid chromatography (UHPLC) multiple-reaction monitoring analysis and were stable under all the tested experimental conditions. Recoveries of the enrichment step from spiked bovine serum albumin digests were >80% for the commercial Fe-IMAC kit and the Strata X-AW sorbent. Shotgun proteomics was used on the same samples to evaluate the selectivity, calculated as the number of coenriched peptides, and it was found to be better for the Fe-IMAC kit. Therefore, the Fe-IMAC protocol was embedded in a shotgun-proteomics workflow and applied to serum spiked with the sulfopeptides before protein dephosphorylation and digestion. The recovery of the entire analytical workflow was 20%, which was compatible with previous data on TiO2 phosphopeptide enrichment. Despite the potential, no sulfopeptide was confidently identified in serum digests by conventional shotgun proteomics, probably due to very low abundance of native sulfoproteins, poor ionization efficiency of sulfopeptides in the positive mode, and lack of unambiguous sulfopeptide identification by bioinformatics software. In this context, the use of negative-ionization mode with high-resolution mass spectrometry appeared promising to improve the sensibility and allow sulfopeptide identification in complex samples.

[more...]

A new opening for the tricky untargeted investigation of natural and modified short peptides

Talanta. 2020. Cerrato, A et al. Sapienza Univ Roma, Dept Chem, Piazzale Aldo Moro 5, I-00185 Rome, Italy.

ABSTRACT: Short peptides are of extreme interest in clinical and food research fields, nevertheless they still represent a crucial analytical issue. The main aim of this paper was the development of an analytical platform for a considerable advancement in short peptides identification. For the first time, short sequences presenting both natural and post-translationally modified amino acids were comprehensively studied thanks to the generation of specific databases. Short peptide databases had a dual purpose.

First, they were employed as inclusion lists for a suspect screening mass-spectrometric analysis, overcoming the limits of data dependent acquisition mode and allowing the fragmentation of such low-abundance substances. Moreover, the databases were implemented in Compound Discoverer 3.0, a software dedicated to the analysis of short molecules, for the creation of a data processing workflow specifically dedicated to short peptide tentative identification. For this purpose, a detailed study of short peptide fragmentation pathways was carried out for the first time. The proposed method was applied to the study of short peptide sequences in enriched urine samples and led to the tentative identification more than 200 short natural and modified short peptides, the highest number ever reported.

[more...]

Identification Of A Novel Histone Derived Antimicrobial Peptide In Airway Surface Liquid.

FASEB JOURNAL. 2020. Biggart, MGS et al. St George’s University Of London

ABSTRACT: The airway surface liquid (ASL), a protective layer secreted by the airway epithelium, represents the first line of defence against inhaled infectious material. It contains a complex array of secreted proteins and peptides that aid the neutralisation and removal of inhaled microbes and toxicants. The ASL also contains many proteases that can cleave proteins to generate further bioactive peptides. We therefore hypothesised that the airway ASL sustained an extensive peptidome containing novel bioactive peptides.

We examined the ASL peptide profile of normal human bronchial epithelial cells transformed with BMI-1 (NHBE-BMI1) and cell-lines Calu3 (submucosal adenocarcinoma) and H441 (Clara cell like adenocarcinoma). ASL was acquired by washing the apical surface of epithelial monolayers grown at air-liquid interface with PBS (100μl) at 0, 24 and 120 hours after exposure to hyperglycaemic (25mM glucose) or normoglycaemic (5mM glucose and 20mM mannitol) basolateral medium. The ASL peptides < 10kDa, were subsequently isolated and analysed using the Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap™ Mass Spectrometer. The resulting spectra of native peptides and their subsequent fragments were analysed using the pNovo De novo sequencing tool. Our preliminary results identified 4765 unique peptides (NHBE-BMI1: 646, Calu3: 3150, H441: 1197). Of these, 37 peptides were common to all samples regardless of glycaemic state. A number of histone derived peptides were identified in all samples and cell lines. These peptides had a proline-alanine rich N-terminal region and a cationic C-terminal, similar to antimicrobial peptides from histone H1 found in Atlantic salmon skin mucous. Synthetic peptides were produced by SPOT synthesis and the antimicrobial activity tested against luminescent Pseudomonas aeruginosa over 18 hours at 37°C in (100mM Tris (pH 7) and 20mM glucose). One of the crude peptides elicited a dose dependent inhibition of bacterial luminescence at 4.63±1.34 μM (n=3), indicating no bacteria survived. Truncated variants of this peptide containing the alanine-proline rich N-terminal sequence or the cationic C-terminal sequence were produced to determine if a specific region of the peptide was required for antimicrobial activity. The N and the C terminal peptides had their antimicrobial activity significantly decreased, only reducing bacterial luminescence by 28.06±2.27% and 35.28±9.45% respectively compared to the full-length peptide; 99.99±0.001% (all normalised to untreated bacterial luminescence, n=4, p<0.0001, ±SD). This indicates that both regions are required for antimicrobial activity. An immunoblot of concentrated native ASL peptides and cell lysates showed the presence of known histone protein epitopes in our peptide of interest. Furthermore, we identified this histone-derived peptide in sputum samples from both Cystic Fibrosis and normal healthy patients. Our preliminary data indicates that this novel histone derived peptide may serve an antimicrobial function in the ASL.

[more...]

Identifying sialylation linkages at the glycopeptide level by glycosyltransferase labeling assisted mass spectrometry (GLAMS)

Analytical chemistry. 2020. Zhu, H et al. Georgia State Univ, Dept Chem, Atlanta, GA 30303 USA.

ABSTRACT: Precise assignment of sialylation linkages at the glycopeptide level is of importance in bottom-up glycoproteomics and an indispensable step to understand the function of glycoproteins in pathogen-host interactions and cancer progression. Even though some efforts have been dedicated to the discrimination of alpha 2,3/alpha 2,6-sialylated isomers, unambiguous identification of sialoglycopeptide isomers is still needed. Herein, we developed an innovative glycosyltransferase labeling assisted mass spectrometry (GLAMS) strategy.

After specific enzymatic labeling, oxonium ions from higher-energy C-trap dissociation (HCD) fragmentation of alpha 2,3-sailoglycopeptides then generate unique reporters to distinctly differentiate those of alpha 2,6-sailoglycopeptide isomers. 'With this strategy, a total of 1236 linkage-specific sialoglycopeptides were successfully identified from 161 glycoproteins in human serum.

[more...]

A streamlined pipeline for multiplexed quantitative site-specific N-glycoproteomics

Nature Communications. 2020. Fang, P et al. Max Planck Inst Biophys Chem, Bioanalyt Mass Spectrometry Grp, D-37077 Gottingen, Germany.

ABSTRACT: Regulation of protein N-glycosylation is essential in human cells. However, large-scale, accurate, and site-specific quantification of glycosylation is still technically challenging. We here introduce SugarQuant, an integrated mass spectrometry-based pipeline comprising protein aggregation capture (PAC)-based sample preparation, multi-notch MS3 acquisition (Glyco-SPS-MS3) and a data-processing tool (GlycoBinder) that enables confident identification and quantification of intact glycopeptides in complex biological samples.

PAC significantly reduces sample-handling time without compromising sensitivity. Glyco-SPS-MS3 combines high-resolution MS2 and MS3 scans, resulting in enhanced reporter signals of isobaric mass tags, improved detection of N-glycopeptide fragments, and lowered interference in multiplexed quantification. GlycoBinder enables streamlined processing of Glyco-SPS-MS3 data, followed by a two-step database search, which increases the identification rates of glycopeptides by 22% compared with conventional strategies. We apply SugarQuant to identify and quantify more than 5,000 unique glycoforms in Burkitt's lymphoma cells, and determine site-specific glycosylation changes that occurred upon inhibition of fucosylation at high confidence. Comprehensive quantitative profiling of intact glycopeptides remains technically challenging. To address this, the authors here develop an integrated quantitative glycoproteomic workflow, including optimized sample preparation, multiplexed quantification and a dedicated data processing tool.

[more...]

基于多头注意力机制和残差神经网络的肽谱匹配打分算法

Journal of Computer Applications. 2020. 闵鑫 et al. 山东理工大学计算机科学与技术学院

ABSTRACT: Peptide spectrum match scoring algorithm plays a key role in the peptide sequence identification,and the traditional scoring algorithm cannot effectively make full use of the peptide fragmentation pattern to perform scoring. In order to solve the problem,a multi-classification probability sum scoring algorithm combined with the peptide sequence information representation called deepscore- alpha was proposed. In this algorithm,the second scoring was not performed with the consideration of global information,and there was no limitation on the similarity calculation method of theoretical mass spectrum and experimental mass spectrum.

In the algorithm,a one-dimensional residual network was used to extract the underlying information of the sequence,and then the effects of different peptide bonds on the current peptide bond fracture were integrated through the multi-attention mechanism to generate the final fragmention relative intensity distribution probability matrix,after that,the final peptide spectrum match score was calculated by combining the actual relative intensity of the peptide sequence fragmention. This algorithm was compared with Comet and MSGF+,two common open source identification tools. The results show that when False Discovery Rate(FDR)was 0.01 on humanbody proteome dataset,the number of peptide sequences retained by deepScore-alpha is increased by about 14%,and the Top1 hit ratio(the proportion of the correct peptide sequences in the spectrum with the highest score)of this algorithm is increased by about 5 percentage points. The generalization performance test of the model trained by human ProteomeTools2 dataset show that the number of sequences peptide retained by deepScore- alpha at FDR of 0.01 is improved by about 7%,the Top1 hit ratio of this algorithm is increased by about 5 percentage points,and the identification results from Decoy library in the Top1 is decreased by about 60%. Experimental results prove that,the algorithm can retain more peptide sequences at lower FDR value, improve the Top1 hit ratio,and has good generalization performance.

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Virus-receptor interactions of glycosylated SARS-CoV-2 spike and human ACE2 receptor

Cell host & microbe. 2020. Zhao, P et al. Univ Georgia, Dept Biochem & Mol Biol, Complex Carbohydrate Res Ctr, Athens, GA 30602 USA.

ABSTRACT: The SARS-CoV-2 betacoronavirus uses its highly glycosylated trimeric Spike protein to bind to the cell surface receptor angiotensin converting enzyme 2 (ACE2) glycoprotein and facilitate host cell entry. We utilized glycomics-informed glycoproteomics to characterize site-specific microheterogeneity of glycosylation for a recombinant trimer Spike mimetic immunogen and for a soluble version of human ACE2. We combined this information with bioinformatics analyses of natural variants and with existing 3D structures of both glycoproteins to generate molecular dynamics simulations of each glycoprotein both alone and interacting with one another.

Our results highlight roles for glycans in sterically masking polypeptide epitopes and directly modulating Spike-ACE2 interactions. Furthermore, our results illustrate the impact of viral evolution and divergence on Spike glycosylation, as well as the influence of natural variants on ACE2 receptor glycosylation. Taken together, these data can facilitate immunogen design to achieve antibody neutralization and inform therapeutic strategies to inhibit viral infection.

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Mass spectrometry analysis of newly emerging coronavirus HCoV-19 spike protein and human ACE2 reveals camouflaging glycans and unique post-translational modifications

Engineering (Beijing, China). 2020. Sun, Zeyu et al. State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310011, China

ABSTRACT: The COVID-19 pandemic has led to worldwide efforts to understand the biological traits of the newly identified HCoV-19 virus. In this mass spectrometry (MS)-based study, we reveal that out of 21 possible glycosites in the HCoV-19 S protein, 20 are completely occupied by N-glycans, predominantly of the oligomannose type. All seven glycosylation sites in human angiotensin I converting enzyme 2 (hACE2) were found to be completely occupied, mainly by complex N-glycans. However, glycosylation did not directly contribute to the binding affinity between HCoV-19 S and hACE2.

Additional post-translational modification (PTM) was identified, including multiple methylated sites in both proteins and multiple sites with hydroxylproline in hACE2. Refined structural models of HCoV-19 S and hACE2 were built by adding N-glycan and PTMs to recently published cryogenic electron microscopy (cryo-EM) structures. The PTM and glycan maps of HCoV-19 S and hACE2 provide additional structural details for studying the mechanisms underlying host attachment and the immune response of HCoV-19, as well as knowledge for developing desperately needed remedies and vaccines. © 2020 THE AUTHORS. Published by Elsevier LTD on behalf of Chinese Academy of Engineering and Higher Education Press Limited Company.

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Relative retention time estimation improves N-glycopeptide identifications by LC--MS/MS

Journal of proteome research. 2020. Klein, J et al. Boston Univ, Program Bioinformat, Boston, MA 02215 USA.

ABSTRACT: Glycopeptides identified by tandem mass spectrometry rely on the identification of the peptide backbone sequence and the attached glycan(s) by the incomplete fragmentation of both moieties. This may lead to ambiguous identifications where multiple structures could explain the same spectrum equally well due to missing information in the mass spectrum or incorrect precursor mass determination. To date, approaches to solving these problems have been limited, and few inroads have been made to address these issues.

We present a technique to address some of these challenges and demonstrate it on previously published data sets. We use a linear modeling approach to learn the influence of the glycan composition on the retention time of a glycopeptide and use these models to validate glycopeptides within the same experiment, detecting over 400 incorrect cases during the MS/MS search and correcting 75 cases that could not be identified based on mass alone. We make this technique available as a command line executable program, written in Python and C, freely available at haps://github.com/mobiusklein/glycresoft in source form, with precompiled binaries for Windows.

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SugarPy facilitates the universal, discovery-driven analysis of intact glycopeptides

Bioinformatics. 2020. Schulze, S et al. Univ Penn, Dept Biol, Leidy Labs, Philadelphia, PA 19104 USA.

ABSTRACT: Motivation: Protein glycosylation is a complex post-translational modification with crucial cellular functions in all domains of life. Currently, large-scale glycoproteomics approaches rely on glycan database dependent algorithms and are thus unsuitable for discovery-driven analyses of glycoproteomes. Results: Therefore, we devised SugarPy, a glycan database independent Python module, and validated it on the glycoproteome of human breast milk. We further demonstrated its applicability by analyzing glycoproteomes with uncommon glycans stemming from the green alga Chlamydomonas reinhardtii and the archaeon Haloferax volcanii.

SugarPy also facilitated the novel characterization of glycoproteins from the red alga Cyanidioschyzon merolae.

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Characterization of N-linked intact glycopeptide signatures of plasma IgGs from patients with prostate carcinoma and benign prostatic hyperplasia for diagnosis pre-stratification

Analyst. 2020. Zhang, Y et al. Sichuan Univ, West China Hosp, MOH, Key Lab Transplant Engn & Immunol, Chengdu 610041, Peoples R China.

ABSTRACT: The discovery of novel non-invasive biomarkers for discriminating between prostate carcinoma (PCa) patients and benign prostatic hyperplasia (BPH) patients is necessary to reduce the burden of biopsies, avoid overdiagnosis and improve quality of life. Previous studies suggest that abnormal glycosylation of immunoglobulin gamma molecules (IgGs) is strongly associated with immunological diseases and prostate diseases. Hence, characterizing N-linked intact glycopeptides of IgGs that correspond to theN-glycan structure with specific site information might enable a better understanding of the molecular pathogenesis and discovery of novel signatures in preoperative discrimination of BPH from PCa.

In this study, we profiled N-linked intact glycopeptides of purified IgGs from 51 PCa patients and 45 BPH patients by our developedN-glycoproteomic method using hydrophilic interaction liquid chromatography enrichment coupled with high resolution LC-MS/MS. The quantitative analysis of the N-linked intact glycopeptides using pGlyco 2.0 and MaxQuant software provided quantitative information on plasma IgG subclass-specific and site-specificN-glycosylation. As a result, we found four aberrantly expressed N-linked intact glycopeptides across different IgG subclasses. In particular, theN-glycopeptide IgG2-GP09 (EEQFNSTFR (H5N5S1)) was dramatically elevated in plasma from PCa patients, compared with that in BPH patients (PCa/BPH ratio = 5.74,p= 0.001). Additionally, the variations in these N-linked intact glycopeptide abundances were not caused by the changes in the IgG concentrations. Furthermore, IgG2-GP09 displayed a more powerful prediction capability (auROC = 0.702) for distinguishing PCa from BPH than the clinical index t-PSA (auROC = 0.681) when used alone or in combination with other indicators (auROC = 0.853). In conclusion, these abnormally expressed N-linked intact glycopeptides have potential for non-invasive monitoring and pre-stratification of prostate diseases.

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N-glycan structures of target cancer biomarker characterized by two-dimensional gel electrophoresis and mass spectrometry

Analytica Chimica Acta. 2020. Liu, S et al. Shanghai Jiao Tong Univ, Sch Life Sci & Biotechnol, Joint Int Res Lab Metab & Dev Sci, State Key Lab Microbial Metab, Shanghai 200240, Peoples R China.

ABSTRACT: Glycoproteins are important biomarkers for cancers, while most glycoproteomics biomarkers suffering from low sensitivity and specificity due to their uncharacterized glycan structures. AZGP1 is a potential biomarker for salivary diagnostics of lung cancer, which is used as a model glycoprotein in this study for method development. We initially analyzed salivary N-glycoproteome by using lectin affinity chromatography and more than 300 N-glycoproteins were identified, including AZGP1. 7 gel spots of AZGP1 were resolved by two-dimensional gel electrophoresis and further confirmed by two-dimensional western blot as well as mass spectrometry.

The isomeric glycan structures of AZGP1 in these spots were systematically characterized both at composition level and at structure level. Our results revealed 10 glycan compositions for salivary AZGP1, including core fucosylated glycans on Asn128 and sialylated glycans on Asn109 and Asn112. We further compared the glycan structures of salivary AZGP1 from lung cancer group and control group. Accordingly, 14 and 7 potential glycan structures were successfully revealed, respectively. In total, 15 glycan compositions and 22 potential glycan structures were identified and characterized for AZGP1, including some different structures with the same compositions. In particular, 5 potential glycan structures were identified as lung cancer unique signatures. Our developed strategy holds promise for thorough identification of glycan structures on a target glycoprotein biomarker. In-depth characterization of its glycan structures will ultimately enhance its sensitivity and specificity for cancer detection. (C) 2020 Elsevier B.V. All rights reserved.

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Glycomics-informed glycoproteomic analysis of site-specific glycosylation for SARS-CoV-2 spike protein

STAR protocols. 2020. Rosenbalm, Katelyn E et al. Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA

ABSTRACT: This protocol describes an integrated approach for analyzing site-specific N- and O-linked glycosylation of SARS-CoV-2 spike protein by mass spectrometry. Glycoproteomics analyzes intact glycopeptides to examine site-specific microheterogeneity of glycoproteins. Glycomics provides structural characterization on any glycan assignments by glycoproteomics. This procedure can be modified and applied to a variety of N- and/or O-linked glycoproteins. Combined with bioinformatics, the glycomics-informed glycoproteomics may be useful in generating 3D molecular dynamics simulations of certain glycoproteins alone or interacting with one another.

For complete details on the use and execution of this protocol, please refer to Zhao etal. (2020). © 2020 The Author(s).

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Glycoproteomic analysis of human urinary exosomes

Analytical Chemistry. 2020. Brown, CJ et al. Indiana Univ, Dept Chem, Bloomington, IN 47401 USA.

ABSTRACT: Exosomes represent a class of secreted biological vesicles, which have recently gained attention due to their function as intertissue and interorganism transporters of genetic materials, small molecules, lipids, and proteins. Although the protein constituents of these exosomes are often glycosylated, a large-scale characterization of the glycoproteome has not yet been completed. This study identified 3144 unique glycosylation events belonging to 378 glycoproteins and 604 unique protein sites of glycosylation.

With these data, we investigated the level of glycan microheterogeneity within the urinary exosomes, finding on average 5.9 glycans per site. The glycan family abundance on individual proteins showed subtle differences, providing an additional level of molecular characterization compared to the unmodified proteome. Finally, we show protein site-specific changes in regard to the common urinary glycoprotein, uromodulin. While uromodulin is an individual case, these same site-specific analyses provide a way forward for developing diagnostic glycoprotein biomarkers with urine as a noninvasive biological fluid. This study represents an important first step in understanding the functional urinary glycoproteome.

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An ultrafast and highly efficient enrichment method for both N-Glycopeptides and N-Glycans by bacterial cellulose

Analytica Chimica Acta. 2020. Wu, MX et al. Fudan Univ, Dept Chem, Shanghai 200032, Peoples R China.

ABSTRACT: A powerful and fast glycopeptide/glycan enrichment method is critical for the efficiency and throughput of mass spectrometry (MS)-based glycoproteomic and glycomic analyses, especially for large-scale sample analysis. Here, we report an ultrafast and effective method for both intact N-glycopeptide and N-glycan enrichment and apply it to human serum samples. In this method, a natural hydrophilic material, bacterial cellulose (BC), was adopted and fully optimized for enrichment. This method offers the following advantages: (i) The enrichment material has natural hydrophilicity and is low-cost, biocompatible, biodegradable and easily accessible; (ii) the whole enrichment procedure is remarkably simple and fast.

It takes only 10 min for intact glycopeptides/glycans to be easily purified from mixtures; (iii) the specificity of this method is over 94% for both glycan and glycopeptide enrichment; and (iv) the outstanding specificity of this technique enables high isolation efficiency for the enrichment of both intact glycopeptides and glycans. A total of 36 N-glycans and 31 N-glycopeptides were identified from human immunoglobulin G (IgG). The glycan and glycopeptide absorption capacity of BC was as high as 333 mg/mg and 250 mg/mg (IgG/BC) respectively. The selectivity for glycan and glycopeptide enrichment reached 1:100 (IgG/bovine serum albumin (BSA), molar ratio) and 1:200 (maltoheptaose (DP7)/BSA, molar ratio), respectively. Furthermore, a total of 159 N-glycans and 523 N-glycopeptides were identified in human serum by using this method. Overall, the BC-based enrichment method we present here provides an ultrafast and highly efficient method for the enrichment of both N-glycopeptides and N glycans in complex samples and shows great potential in large-scale glycoproteomic and glycomic analyses. (c) 2020 Published by Elsevier B.V.

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One-step synthesis of hydrophilic microspheres for highly selective enrichment of N-linked glycopeptides

Analytica Chimica Acta. 2020. Zhang, N et al. Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China.

ABSTRACT: A polyacrylamide-based hydrophilic microsphere with a lot of hydroxyl groups on surface (PAM-OH HMS) was prepared in one step. The synthetic process was simple reverse suspension polymerization without any chemical derivation or grafting steps. The properties of obtained HMS were characterized by scanning electron microscopy (SEM), static water contact angle measurement, and FT-IR. The abundant hydroxyl groups on the surface make the material highly good hydrophilic and thus it was utilized for N-glycopeptides enrichment.

The enrichment efficiency of PAM-OH HMSs was demonstrated by capturing N-linked glycopeptides from tryptic digest of human immunoglobulin G (IgG). The detection sensitivity for N-glycopeptides identification by MALDI-TOF MS was as low as 10 fmol for tryptic digest of standard human IgG. The selectivity of the HMS towards N-glycopeptides had almost no decrease when the molar ratio of BSA tryptic digest to IgG tryptic digest was increased from 10:1 to 100:1. Moreover, in the LC-MS/MS analysis of real biological sample, a total of 344 unique N-glycosites in 598 unique N-glycopeptides from 172 N-glycoproteins were identified from 2 mu L human serum after deglycosylated by PNGase F, and 825 intact N-glycopeptides with different types of glycoform were detected when directly analyzed the N-glycopeptides enriched by PAM-OH HMS. To show the potential of our material in solving real biological issues, N-glycopeptides in the serum from hepatocelluar carcinoma (HCC) patient and health control were enriched and quantified. All the experiments demonstrated that this polyacrylamide-based hydrophilic microsphere shows a great potential to enrich the low-abundance N-glycopeptides for glycoproteome analysis of real complicated biological samples. (C) 2020 Elsevier B.V. All rights reserved.

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Computational classification of core and outer fucosylation of N-glycoproteins in human plasma using collision-induced dissociation in mass spectrometry

Rapid Communications in Mass Spectrometry. 2020. Jeong, HK et al. Korea Basic Sci Inst, Res Ctr Bioconvergence Anal, 162 Yeongudanji Ro, Cheongju 28119, South Korea.

ABSTRACT: Rationale Glycoprotein fucosylation, one of the major posttranslational modifications, is known to be highly involved in proteins related to various cancers. Fucosylation occurs in the core and/or outer sites of N-glycopeptides. Elucidation of the fucosylation type of N-glycoproteins is therefore important. However, it has remained a challenge to classify the fucosylation types of N-glycopeptides using collision-induced dissociation (CID) tandem mass (MS/MS) spectra. Methods The relative intensities of the Y1F, Y2F, Y3F, and Y4F product ions in the CID-MS/MS spectra of the IgG N-glycopeptides were measured for core fucosylation.

The Core Fucose Index (CFI) was then calculated by multiplication of the relative intensities with a weight factor from logistic regression to differentiate between the core and none fucosylation. From the relative intensities of the B2F and B3SF ions of the MS/MS spectra of the AGP N-glycopeptides for outer fucosylation, the Outer Fucose Index (OFI) was calculated to differentiate between the outer and none fucosylation. Results In order to classify core and/or outer fucosylation of N-glycoproteins, we defined the fucosylation score (F-score) by a sigmoidal equation using a combination of the CFI and the OFI. For application, we classified the fucosylation types of N-glycoproteins in human plasma with 99.7% accuracy from the F-score. Human plasma samples showed 54.4%, 33.3%, 10.3%, and 1.6% for none, core, outer, and dual fucosylated N-glycopeptides, respectively. Core fucosylation was abundant at mono- and bi-antennary N-glycopeptides. Outer fucosylation was abundant at tri- and tetra-antennary N-glycopeptides. In total, 113 N-glycopeptides of 29 glycoproteins from 3365 glycopeptide spectral matches (GPSMs) were classified for different types of fucosylation. Conclusions We established an F-score to classify three different fucosylation types: core, outer, and dual types of N-glycopeptides. The fucosylation types of 20 new N-glycopeptides from 11 glycoproteins in human plasma were classified using the F-score. Therefore, the F-score can be useful for the automatic classification of different types of fucosylation in N-glycoproteins of biological fluids including plasma, serum, and urine.

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Peptide release after simulated infant in vitro digestion of dry heated cow’s milk protein and transport of potentially immunoreactive peptides across the Caco-2 cell monolayer

Nutrients. 2020. Zenker, HE et al. Wageningen Univ & Res Ctr, Food Qual & Design Grp, NL-6708 WG Wageningen, Netherlands.

ABSTRACT: Dry heating of cow's milk protein, as applied in the production of "baked milk", facilitates the resolution of cow's milk allergy symptoms upon digestion. The heating and glycation-induced changes of the protein structure can affect both digestibility and immunoreactivity. The immunological consequences may be due to changes in the peptide profile of the digested dry heated milk protein. Therefore, cow's milk protein powder was heated at low temperature (60 degrees C) and high temperature (130 degrees C) and applied to simulated infant in vitro digestion.

Digestion-derived peptides after 10 min and 60 min in the intestinal phase were measured using LC-MS/MS. Moreover, digests after 10 min intestinal digestion were applied to a Caco-2 cell monolayer. T-cell epitopes were analysed using prediction software, while specific immunoglobin E (sIgE) binding epitopes were identified based on the existing literature. The largest number of sIgE binding epitopes was found in unheated samples, while T-cell epitopes were equally represented in all samples. Transport of glycated peptide indicated a preference for glucosyl lysine and lactosyl-lysine-modified peptides, while transport of peptides containing epitope structures was limited. This showed that the release of immunoreactive peptides can be affected by the applied heating conditions; however, availability of peptides containing epitopes might be limited.

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Quantitative analysis of $\alpha$-1-antitrypsin glycosylation isoforms in HCC patients using LC-HCD-PRM-MS

Analytical Chemistry. 2020. Yin, HD et al. Hong Kong Polytech Univ, Shenzhen Res Inst, Shenzhen, Peoples R China.

ABSTRACT: The change in glycosylation of serum proteins is often associated with the development of various diseases and thus can be used for diagnosis. In this study, a liquid chromatography-tandem mass spectrometry-based method is used for accurate structural analysis and quantification of site-specific glycoforms of serum alpha-1-antitrypsin (A1AT) in early-stage HCC and cirrhosis patients. Serum protein A1AT was purified from patient sera by immunoprecipitation with anti-A1AT antibody conjugated agarose beads, and the isolated A1AT protein was digested and analyzed by LC-MS/MS.

Two tandem mass spectrometry strategies are integrated in this study: a nontargeted stepped HCD strategy for structural analysis of A1AT glycopeptides and a targeted parallel reaction monitoring (PRM) strategy for quantification of site-specific glycoforms of A1AT in HCC and cirrhosis patient sera. Accordingly, pGlyco2.0 software was used for glycopeptide identification, and Skyline software was used for glycoform quantification using the Y1 ion (peptide+GlcNAc) in MS/MS spectra. Ten site-specific glycopeptides of A1AT were identified with stepped HCD-MS/MS in patient samples, 7 of which were further quantified using HCD-PRM-MS among patient samples. We found that our strategy was able to distinguish isomers of glycopeptides where several isomers showed distinctly different patterns between cirrhosis and HCC patients. We also found that the ratio of different charge states (2+/3+) of one glycopeptide of A1AT can significantly discriminate early-stage HCC from cirrhosis with the area under the receiver operating characteristic curve AUC of 0.9. Further analysis showed that the difference may be related to the sialic acid/galactose linkage of the glycan motif.

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Quantitative analysis of global protein stability rates in tissues

Scientific reports. 2020. McClatchy, DB et al. Scripps Res Inst, Dept Mol Med, La Jolla, CA 92037 USA.

ABSTRACT: Protein degradation is an essential mechanism for maintaining proteostasis in response to internal and external perturbations. Disruption of this process is implicated in many human diseases. We present a new technique, QUAD (Quantification of Azidohomoalanine Degradation), to analyze the global degradation rates in tissues using a non-canonical amino acid and mass spectrometry. QUAD analysis reveals that protein stability varied within tissues, but discernible trends in the data suggest that cellular environment is a major factor dictating stability.

Within a tissue, different organelles and protein functions were enriched with different stability patterns. QUAD analysis demonstrated that protein stability is enhanced with age in the brain but not in the liver. Overall, QUAD allows the first global quantitation of protein stability rates in tissues, which will allow new insights and hypotheses in basic and translational research.

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Temporal Quantitative Profiling of Newly Synthesized Proteins during A$\beta$ Accumulation

Journal of Proteome Research. 2020. Ma, YH et al. Scripps Res Inst, Dept Chem Physiol & Mol & Cellular Neurobiol, La Jolla, CA 92037 USA.

ABSTRACT: Accumulation of aggregated amyloid beta (A beta) in the brain is believed to impair multiple cellular pathways and play a central role in Alzheimer's disease pathology. However, how this process is regulated remains unclear. In theory, measuring protein synthesis is the most direct way to evaluate a cell's response to stimuli, but to date, there have been few reliable methods to do this. To identify the protein regulatory network during the development of A beta deposition in AD, we applied a new proteomic technique to quantitate newly synthesized protein (NSP) changes in the cerebral cortex and hippocampus of 2-, 5-, and 9-month-old APP/PS1 AD transgenic mice.

This bio-orthogonal noncanonical amino acid tagging analysis combined PALM (pulse azidohomoalanine labeling in mammals) and HILAQ (heavy isotope labeled AHA quantitation) to reveal a comprehensive dataset of NSPs prior to and post A beta deposition, including the identification of proteins not previously associated with AD, and demonstrated that the pattern of differentially expressed NSPs is age-dependent. We also found dysregulated vesicle transportation networks including endosomal subunits, coat protein complex I (COPI), and mitochondrial respiratory chain throughout all time points and two brain regions. These results point to a pathological dysregulation of vesicle transportation which occurs prior to A beta accumulation and the onset of AD symptoms, which may progressively impact the entire protein network and thereby drive neurodegeneration. This study illustrates key pathway regulation responses to the development of AD pathogenesis by directly measuring the changes in protein synthesis and provides unique insights into the mechanisms that underlie AD.

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MASH explorer: a universal software environment for top-down proteomics

Journal of proteome research. 2020. Wu, Zhijie et al. Univ Wisconsin, Dept Biostat & Med Informat, Madison, WI 53705 USA; Univ Wisconsin, Carbone Canc Ctr, Madison, WI 53705 USA; Univ Wisconsin, Sch Med & Publ Hlth, Dept Cell & Regenerat Biol, Dept Chem, Madison, WI 53705 USA; Univ Wisconsin, Sch Med & Publ Hlth, Human Prote Program, Madison, WI 53705 USA

ABSTRACT: Top-down mass spectrometry (MS)-based proteomics enable a comprehensive analysis of proteoforms with molecular specificity to achieve a proteome-wide understanding of protein functions. However, the lack of a universal software for top-down proteomics is becoming increasingly recognized as a major barrier, especially for newcomers. Here, we have developed MASH Explorer, a universal, comprehensive, and user-friendly software environment for top-down proteomics. MASH Explorer integrates multiple spectral deconvolution and database search algorithms into a single, universal platform which can process top-down proteomics data from various vendor formats, for the first time.

It addresses the urgent need in the rapidly growing top-down proteomics community and is freely available to all users worldwide. With the critical need and tremendous support from the community, we envision that this MASH Explorer software package will play an integral role in advancing top-down proteomics to realize its full potential for biomedical research.

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In silico spectral libraries by deep learning facilitate data-independent acquisition proteomics

Nature communications. 2020. Yang, Y et al. Fudan Univ, Dept Chem, Shanghai Stomatol Hosp, Shanghai 200000, Peoples R China.

ABSTRACT: Data-independent acquisition (DIA) is an emerging technology for quantitative proteomic analysis of large cohorts of samples. However, sample-specific spectral libraries built by data-dependent acquisition (DDA) experiments are required prior to DIA analysis, which is time-consuming and limits the identification/quantification by DIA to the peptides identified by DDA. Herein, we propose DeepDIA, a deep learning-based approach to generate in silico spectral libraries for DIA analysis. We demonstrate that the quality of in silico libraries predicted by instrument-specific models using DeepDIA is comparable to that of experimental libraries, and outperforms libraries generated by global models.

With peptide detectability prediction, in silico libraries can be built directly from protein sequence databases. We further illustrate that DeepDIA can break through the limitation of DDA on peptide/protein detection, and enhance DIA analysis on human serum samples compared to the state-of-the-art protocol using a DDA library. We expect this work expanding the toolbox for DIA proteomics.

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Full-spectrum prediction of peptides tandem mass spectra using deep neural network

Analytical chemistry. 2020. Liu, KY et al. Indiana Univ, Sch Informat Comp & Engn, Bloomington, IN 47405 USA.

ABSTRACT: The ability to predict tandem mass (MS/MS) spectra from peptide sequences can significantly enhance our understanding of the peptide fragmentation process and could improve peptide identification in proteomics. However, current approaches for predicting high-energy collisional dissociation (HCD) spectra are limited to predict the intensities of expected ion types, that is, the a/b/c/x/y/z ions and their neutral loss derivatives (referred to as backbone ions). In practice, backbone ions only account for <70% of total ion intensities in HCD spectra, indicating many intense ions are ignored by current predictors.

In this paper, we present a deep learning approach that can predict the complete spectra (both backbone and nonbackbone ions) directly from peptide sequences. We made no assumptions or expectations on which kind of ions to predict but instead predicting the intensities for all possible m/z. Training this model needs no annotations of fragment ion nor any prior knowledge of the fragmentation rules. Our analyses show that the predicted 2+ and 3+ HCD spectra are highly similar to the experimental spectra, with average full-spectrum cosine similarities of 0.820 (+/- 0.088) and 0.786 (+/- 0.085), respectively, very close to the similarities between the experimental replicated spectra. In contrast, the best-performed backbone only models can only achieve an average similarity below 0.75 and 0.70 for 2+ and 3+ spectra, respectively. Furthermore, we developed a multitask learning (MTL) approach for predicting spectra of insufficient training samples, which allows our model to make accurate predictions for electron transfer dissociation (ETD) spectra and HCD spectra of less abundant charges (1+ and 4+).

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DeepRescore: leveraging deep learning to improve peptide identification in immunopeptidomics

Proteomics. 2020. Li, K et al. Baylor Coll Med, Lester & Sue Smith Breast Ctr, Houston, TX 77030 USA.

ABSTRACT: The identification of major histocompatibility complex (MHC)-binding peptides in mass spectrometry (MS)-based immunopeptideomics relies largely on database search engines developed for proteomics data analysis. However, because immunopeptidomics experiments do not involve enzymatic digestion at specific residues, an inflated search space leads to a high false positive rate and low sensitivity in peptide identification. In order to improve the sensitivity and reliability of peptide identification, a post-processing tool named DeepRescore is developed.

DeepRescore combines peptide features derived from deep learning predictions, namely accurate retention timeand MS/MS spectra predictions, with previously used features to rescore peptide-spectrum matches. Using two public immunopeptidomics datasets, it is shown that rescoring by DeepRescore increases both the sensitivity and reliability of MHC-binding peptide and neoantigen identifications compared to existing methods. It is also shown that the performance improvement is, to a large extent, driven by the deep learning-derived features. DeepRescore is developed using NextFlow and Docker and is available at .

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Hybrid spectral library combining DIA-MS data and a targeted virtual library substantially deepens the proteome coverage

IScience. 2020. Lou, RH et al. ShanghaiTech Univ, iHuman Inst, Shanghai 201210, Peoples R China.

ABSTRACT: Data-independent acquisition mass spectrometry (DIA-MS) is a powerful technique that enables relatively deep proteomic profiling with superior quantification reproducibility. DIA data mining predominantly relies on a spectral library of sufficient proteome coverage that, in most cases, is built on data-dependent acquisition-based analysis of the same sample. To expand the proteome coverage for a pre-determined protein family, we report herein on the construction of a hybrid spectral library that supplements a DIA experiment-derived library with a protein family-targeted virtual library predicted by deep learning.

Leveraging this DIA hybrid library substantially deepens the coverage of three transmembrane protein families (G protein-coupled receptors, ion channels, and transporters) in mouse brain tissues with increases in protein identification of 37%-87% and peptide identification of 58%-161%. Moreover, of the 412 novel GPCR peptides exclusively identified with the DIA hybrid library strategy, 53.6% were validated as present in mouse brain tissues based on orthogonal experimental measurement.

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2019