Building ProteomeTools based on a complete synthetic human proteome

Zolg, Daniel Paul; Wilhelm, Mathias; Schnatbaum, Karsten; Zerweck, Johannes; Knaute, Tobias; Delanghe, Bernard; Bailey, Derek J.; Gessulat, Siegfried; Ehrlich, Hans‐Christian; Weininger, Maximilian; Yu, Ping; Schlegl, Judith; Kramer, Karl; Schmidt, Tobias; Kusebauch, Ulrike; Deutsch, Eric W.; Aebersold, Ruedi; Moritz, Robert L.; Wenschuh, Holger; Moehring, Thomas J.; Aiche, Stephan; Hühmer, Andreas; Reimer, Ulf; Küster, Bernhard

doi:10.1038/nmeth.4153

Cited by 215 publications

(294 citation statements)

References 23 publications

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“…To evaluate the performance of MS2‐Deisotoper, we analyzed 696692 high‐quality, annotated MS/MS spectra of human synthetic peptides generated as part of the ProteomeTools Project . These spectra serve as “ground truth” data as the sequence and thus fragment ions are known for each peptide analyzed.…”

Section: Resultsmentioning

confidence: 99%

“…The human synthetic peptide spectral library that was created using data from ProteomeXchange with the dataset identifier PXD004732, was obtained via the FTP server of the NIST Mass Spectrometry Data Center. It contains 696692 high‐quality, annotated MS/MS spectra generated as part of the ProteomeTools Project . Proteomic SILAC data for human K562 cells from Hamey et al .…”

Section: Methodsmentioning

confidence: 99%

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MS2‐Deisotoper: A Tool for Deisotoping High‐Resolution MS/MS Spectra in Normal and Heavy Isotope‐Labelled Samples

et al. 2019

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High‐resolution MS/MS spectra of peptides can be deisotoped to identify monoisotopic masses of peptide fragments. The use of such masses should improve protein identification rates. However, deisotoping is not universally used and its benefits have not been fully explored. Here, MS2‐Deisotoper, a tool for use prior to database search, is used to identify monoisotopic peaks in centroided MS/MS spectra. MS2‐Deisotoper works by comparing the mass and relative intensity of each peptide fragment peak to every other peak of greater mass, and by applying a set of rules concerning mass and intensity differences. After comprehensive parameter optimization, it is shown that MS2‐Deisotoper can improve the number of peptide spectrum matches (PSMs) identified by up to 8.2% and proteins by up to 2.8%. It is effective with SILAC and non‐SILAC MS/MS data. The identification of unique peptide sequences is also improved, increasing the number of human proteoforms by 3.7%. Detailed investigation of results shows that deisotoping increases Mascot ion scores, improves FDR estimation for PSMs, and leads to greater protein sequence coverage. At a peptide level, it is found that the efficacy of deisotoping is affected by peptide mass and charge. MS2‐Deisotoper can be used via a user interface or as a command‐line tool.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

MS2‐Deisotoper: A Tool for Deisotoping High‐Resolution MS/MS Spectra in Normal and Heavy Isotope‐Labelled Samples

et al. 2019

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“…All synthetic peptide pools were analyzed by nanoLC-MS/MS on an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) as previously described (28). Briefly, each peptide pool was measured using a 'survey method' consisting of an Orbitrap full MS scan (60,000 resolution, were processed by Mascot distiller and all .raw files were searched by Mascot using the same parameters, database, and acceptance criteria indicated above for the human tissue data.…”

Section: Lc-ms/ms Measurement and Data Analysis Of Synthetic Peptidesmentioning

confidence: 99%

Mining the Human Tissue Proteome for Protein Citrullination

Lee

Wang

Wilhelm

et al. 2018

Molecular & Cellular Proteomics

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175

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SummaryCitrullination is a post-translational modification of arginine catalyzed by five peptidylarginine deiminases (PADs) in humans. The loss of a positive charge may cause structural or functional alterations and while the modification has been linked to several diseases including rheumatoid arthritis and cancer, its physiological or pathophysiological roles remain largely unclear. In part this is owing to limitations in available methodology able to robustly enrich, detect and localize the modification. As a result, only few citrullination sites have been identified on human proteins with high confidence. In this study, we mined data from mass spectrometry-based deep proteomic profiling of 30 human tissues to identify citrullination sites on endogenous proteins. Database searching of ~70 million tandem mass spectra yielded ~13,000 candidate spectra which were further triaged by spectrum quality metrics and the detection of the specific neutral loss of isocyanic acid from citrullinated peptides to reduce false positives. Because citrullination is easily confused with deamidation, we synthetized ~2,200 citrullinated and 1,300 deamidated peptides to build a library of reference spectra. This led to the validation of 375 citrullination sites on 209 human proteins. Further analysis showed that >80% of the identified modifications sites were new and for 56% of the proteins, citrullination was detected for the first time. Sequence motif analysis revealed a strong preference for Asp and Gly, residues around the citrullination site. Interestingly, while the modification was detected in 26 human tissues with the highest levels found in brain and lung, citrullination levels did not correlate well with protein expression of the PAD enzymes.Even though the current work represents the largest survey of protein citrullination to date, the modification was mostly detected on high abundant proteins arguing that the development of specific enrichment methods would be required in order to study the full extent of cellular protein citrullination.

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“…LC solvent A was 5% DMSO, 0.1% formic acid in ultra-pure water, LC solvent B was 5% DMSO, 0.1% formic acid in acetonitrile (19). Peptide pools were measured as previously described (16).…”

Section: Lc-ms/ms Analysismentioning

confidence: 99%