pFind Studio: a computational solution for mass spectrometry-based proteomics
2019
Journal of the American Chemical Society2019. Resnick, E et al.
Weizmann Inst Sci, Nancy & Stephen Grand Israel Natl Ctr Personalize, Dept Organ Chem, IL-7610001 Rehovot, Israel.
ABSTRACT:Covalent probes can display unmatched potency, selectivity, and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered nonselective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against 10 cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. In contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery.
Use: pFind
Antioxidants & redox signaling2019. Fu, L et al.
Beijing Inst Life, Natl Ctr Prot Sci Beijing, State Key Lab Prote, Beijing Proteome Res Ctr, Beijing 102206, Peoples R China.
ABSTRACT:Aims: Cysteine persulfidation (also called sulfhydration or sulfuration) has emerged as a potential redox mechanism to regulate protein functions and diverse biological processes in hydrogen sulfide (H2S) signaling. Due to its intrinsically unstable nature, working with this modification has proven to be challenging. Although methodological progress has expanded the inventory of persulfidated proteins, there is a continued need to develop methods that can directly and unequivocally identify persulfidated cysteine residues in complex proteomes. Results: A quantitative chemoproteomic method termed as low-pH quantitative thiol reactivity profiling (QTRP) was developed to enable direct site-specific mapping and reactivity profiling of proteomic persulfides and thiols in parallel. The method was first applied to cell lysates treated with NaHS, resulting in the identification of overall 1547 persulfidated sites on 994 proteins. Structural analysis uncovered unique consensus motifs that might define this distinct type of modification. Moreover, the method was extended to profile endogenous protein persulfides in cells expressing H2S-generating enzyme, mouse tissues, and human serum, which led to additional insights into mechanistic, structural, and functional features of persulfidation events, particularly on human serum albumin. Innovation and Conclusion: Low-pH QTRP represents the first method that enables direct and unbiased proteomic mapping of cysteine persulfidation. Our method allows to generate the most comprehensive inventory of persulfidated targets of NaHS so far and to perform the first analysis of in vivo persulfidation events, providing a valuable tool to dissect the biological functions of this important modification. Antioxid. Redox Signal. 00, 000-000.
Use: pFind; pQuant
Cell Chemical Biology2019. Griswold, AR et al.
Mem Sloan Kettering Canc Ctr, Weill Cornell Grad Sch Med Sci, Pharmacol Program, New York, NY 10065 USA.
ABSTRACT:The dipeptidyl peptidases (DPPs) regulate hormones, cytokines, and neuropeptides by cleaving dipeptides after proline from their amino termini. Due to technical challenges, many DPP substrates remain unknown. Here, we introduce a simple method, termed CHOPS (chemical enrichment of protease substrates), for the discovery of protease substrates. CHOPS exploits a 2-pyridinecarboxaldehyde (2PCA)-biotin probe, which selectively biotinylates protein N-termini except those with proline in the second position. CHOPS can, in theory, discover substrates for any protease, but is particularly well suited to discover canonical DPP substrates, as cleaved but not intact DPP substrates can be identified by gel electrophoresis or mass spectrometry. Using CHOPS, we show that DPP8 and DPP9, enzymes that control the Nlrp1 inflammasome through an unknown mechanism, do not directly cleave Nlrp1. We further show that DPP9 robustly cleaves short peptides but not full-length proteins. More generally, this work delineates a practical technology for identifying protease substrates, which we anticipate will complement available "N-terminomic" approaches.
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EMBO JOURNAL2019. Zhang, G et al.
Univ Copenhagen, Novo Nordisk Fdn Ctr Prot Res, Fac Hlth & Med Sci, Copenhagen, Denmark.
ABSTRACT:Kinetochore localized Mad1 is essential for generating a "wait anaphase" signal during mitosis, hereby ensuring accurate chromosome segregation. Inconsistent models for the function and quantitative contribution of the two mammalian Mad1 kinetochore receptors: Bub1 and the Rod-Zw10-Zwilch (RZZ) complex exist. By combining genome editing and RNAi, we achieve penetrant removal of Bub1 and Rod in human cells, which reveals that efficient checkpoint signaling depends on the integrated activities of these proteins. Rod removal reduces the proximity of Bub1 and Mad1, and we can bypass the requirement for Rod by tethering Mad1 to kinetochores or increasing the strength of the Bub1-Mad1 interaction. We find that Bub1 has checkpoint functions independent of Mad1 localization that are supported by low levels of Bub1 suggesting a catalytic function. In conclusion, our results support an integrated model for the Mad1 receptors in which the primary role of RZZ is to localize Mad1 at kinetochores to generate the Mad1-Bub1 complex.
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Technological Innovation and Resources2019. Hao Yang; Yan-Chang Li; Ming-Zhi Zhao et al.
State Key Laboratory of Proteomics; Beijing Proteome Research Center; National Center for Protein Sciences Beijing; Beijing Institute of Lifeomics, Beijing 102206, China;Key Laboratory of Combinatorial Biosynthesis and Drug Discovery of Ministry of Education (Wuhan University), Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China;College of Life Sciences, Hebei University, Baoding 071002, China(X.P.); Key Lab of Intelligent Information Processing of Chinese Academy of Sciences (CAS), Institute of Computing Technology, CAS; University of Chinese Academy of Sciences; Institute of Computing Technology, CAS, Beijing 100190, China
ABSTRACT:De novo peptide sequencing for large-scale proteomics remains challenging because of the lack of full coverage of ion series in tandem mass spectra. We developed a mirror protease of trypsin, acetylated LysargiNase (Ac-LysargiNase), with superior activity and stability. The mirror spectrum pairs derived from the Ac-LysargiNase and trypsin treated samples can generate full b and y ion series, which provide mutual complementarity of each other, and allow us to develop a novel algorithm, pNovoM, for de novo sequencing. Using pNovoM to sequence peptides of purified proteins, the accuracy of the sequence was close to 100%. More importantly, from a large-scale yeast proteome sample digested with trypsin and Ac-LysargiNase individually, 48% of all tandem mass spectra formed mirror spectrum pairs, 97% of which contained full coverage of ion series, resulting in precision de novo sequencing of full-length peptides by pNovoM. This enabled pNovoM to successfully sequence 21,249 peptides from 3,753 proteins and interpreted 44-152% more spectra than pNovo+ and PEAKS at a 5% FDR at the spectrum level. Moreover, the mirror protease strategy had an obvious advantage in sequencing long peptides. We believe that the combination of mirror protease strategy and pNovoM will be an effective approach for precision de novo sequencing on both single proteins and proteome samples.
Use: pFind; pNovo
Journal of the American Society for Mass Spectrometry2019. Li, WX et al.
Yale Univ, Yale Canc Biol Inst, West Haven, CT 06516 USA.
ABSTRACT:Due to the technical advances of mass spectrometers, particularly increased scanning speed and higher MS/MS resolution, the use of data-independent acquisition mass spectrometry (DIA-MS) became more popular, which enables high reproducibility in both proteomic identification and quantification. The current DIA-MS methods normally cover a wide mass range, with the aim to target and identify as many peptides and proteins as possible and therefore frequently generate MS/MS spectra of high complexity. In this report, we assessed the performance and benefits of using small windows with, e.g., 5-m/z width across the peptide elution time. We further devised a new DIA method named RTwinDIA that schedules the small isolation windows in different retention time blocks, taking advantage of the fact that larger peptides are normally eluting later in reversed phase chromatography. We assessed the direct proteomic identification by using shotgun database searching tools such as MaxQuant and pFind, and also Spectronaut with an external comprehensive spectral library of human proteins. We conclude that algorithms like pFind have potential in directly analyzing DIA data acquired with small windows, and that the instrumental time and DIA cycle time, if prioritized to be spent on small windows rather than on covering a broad mass range by large windows, will improve the direct proteome coverage for new biological samples and increase the quantitative precision. These results further provide perspectives for the future convergence between DDA and DIA on faster MS analyzers.
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Journal of proteome research2019. Sun, JS et al.
Sun Yat Sen Univ, Sch Life Sci, State Key Lab Biocontrol, Guangzhou 510275, Guangdong, Peoples R China.
ABSTRACT:In recent years, high-throughput technologies have contributed to the development of a more precise picture of the human proteome. However, 2129 proteins remain listed as missing proteins (MPs) in the newest neXtProt release (2019-02). The main reasons for MPs are a low abundance, a low molecular weight, unexpected modifications, membrane characteristics, and so on. Moreover, >50% of the MS/MS data have not been successfully identified in shotgun proteomics. Open-pFind, an efficient open search engine, recently released by the pFind group in China, might provide an opportunity to identify these buried MPs in complex samples. In this study, proteins and potential MPs were identified using Open-pFind and three other search engines to compare their performance and efficiency with three large-scale data sets digested by three enzymes (Glu-C, Lys-C, and trypsin) with specificity on different amino acid (AA) residues. Our results demonstrated that Open-pFind identified 44.7-93.1% more peptide-spectrum matches and 21.3-61.6% more peptide sequences than the second-best search engine. As a result, Open-pFind detected 53.1% more MP candidates than MaxQuant and 8.8% more candidate MPs than Proteome Discoverer. In total, 5 (PE2) of the 124 MP candidates identified by Open-pFind were verified with 2 or 3 unique peptides containing more than 9 AAs by using a spectrum theoretical prediction with pDeep and synthesized peptide matching with pBuild after spectrum quality analysis, isobaric post-translational modification, and single amino acid variant filtering. These five verified MPs can be saved as PEI proteins. In addition, three other MP candidates were verified with two unique peptides (one peptide containing more than 9 AAs and the other containing only 8 AAs), which was slightly lower than the criteria listed by C-HPP and required additional verification information. More importantly, unexpected modifications were detected in these MPs. All MS data sets have been deposited into ProteomeXchange with the identifier PXDO15759.
Use: pFind; pDeep
International Journal of Mass Spectrometry2019. Tabb, DL et al.
Stellenbosch Univ, Dept Biomed Sci, Div Mol Biol & Human Genet, Bioinformat Unit,South African TB Bioinformat Ini, Cape Town, South Africa.
ABSTRACT:The application of database search algorithms with very wide precursor mass tolerances for the "Open Search" paradigm has brought new efforts at post-translational modification discovery in shotgun proteomes. This approach has motivated the acceleration of database search tools by incorporating fragment indexing features. In this report, we compare open searches and sequence tag searches of high-resolution tandem mass spectra to seek a common "palette" of modifications when analyzing multiple formalin-fixed, paraffin-embedded (FFPE) tissues from Thermo Q-Exactive and SCIEX TripleTOF instruments. While open search in MSFragger produced some gains in identified spectra, careful FDR control limited the best result to 24% more spectra than narrow search (worst result: a loss of 9%). Open pFind produced high apparent sensitivity for PSMs, but entrapment sequences hinted that the actual error rate may be higher than reported by the software. Combining sequence tagging, open search, and chemical knowledge, we converged on this set of PTMs for our four FFPE sets: mono- and di-methylation (nTerm and Lys), single and double oxidation (Met and Pro), and variable carbamidomethylation (nTerm and Cys). (C) 2019 Elsevier B.V. All rights reserved.
Use: pBuild; pFind; pParse
Analytical and Bioanalytical Chemistry2019. Guan, S et al.
Fudan Univ, Dept Chem, Shanghai 200438, Peoples R China.
ABSTRACT:Exosomes are cell-derived functional microparticles which exist in most body fluids. They carry abundant signaling molecules to transfer information between cells and microenvironment. Research on exosomes' heterogeneity and constitute variations has been a heated topic in recent years. In this work, size-dependent sub-proteome analysis of urinary exosomes was investigated by size exclusion chromatography (SEC) firstly. The particle size of urinary exosomes is distributed in four main ranges naturally. We found out that these fractions contained sub-proteomes with great difference in constitution. In each fraction, 206, 134, 157, and 276 unique proteins were identified by LC-MS/MS. Differential expression of exosomal markers such as TSG101, CD9, CD63, and caveolin-1 was observed in these fractions by western blots. Biological function annotation indicated that the proteins identified in each fraction were involved in different molecular and cellular processes. It is proven that SEC can serve as an efficient analytical tool for exosomes isolation and fractionation. This work provides a new strategy to classify exosomes into sub-populations for comprehensive study of heterogeneous functionalities.
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Analytica Chimica Acta2019. Cai, Y et al.
Fudan Univ, Shanghai Canc Ctr, Shanghai 200032, Peoples R China.
ABSTRACT:For mass spectrometry (MS)-based N-glycoproteomics, selective enrichment of N-glycopeptides prior to MS analysis is a crucial step to reduce sample complexity. Enrichment based on covalent coupling is as an increasingly attractive strategy due to the unbiased and highly specific features. However, most of current covalent coupling reactions for N-glycopeptides enrichment are still limited by long coupling time and harsh coupling conditions. Herein, we developed a thiazolidine formation-based approach for ultrafast and highly efficient solid-phase extraction of N-Glycoproteome. With the use of facile synthesis of Cys-terminated magnetic nanoparticles, the oxidized glycan moieties on glycopeptides could be selectively captured by the beta-amino thiols groups on the surface of magnetic nanoparticles through thiazolidine formation. The coupling could be achieved within 30 min under mild condition, eliminating the addition of toxic catalyst or sample-destroying reducing agent. Also, the great enrichment performance for N-glycopeptides were obtained in terms of sensitivity (low fmol levels), selectivity (extracting N-glycopeptides from the mixture of glycopeptides and non-glycopeptides at a 1:100 molar ratio) and reproducibility (CVs<26%). Finally, this proposed method was successfully demonstrated by analyzing the N-glycoproteome from 2 mu L human serum, which offers an alternative purification method for analysis of N-glycoproteome from complex biological samples. (C) 2019 Elsevier B.V. All rights reserved.
Use: pFind