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An ultra-tolerant database search reveals that a myriad of modified peptides contributes to unassigned spectra in shotgun proteomics

Fewer than half of all tandem mass spectrometry (MS/MS) spectra acquired in shotgun proteomics experiments are typically matched to a peptide with high confidence. Here we determine the identity of unassigned peptides using an ultra-tolerant Sequest database search that allows peptide matching even...

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Detalles Bibliográficos
Autores principales: Chick, Joel M., Kolippakkam, Deepak, Nusinow, David P., Zhai, Bo, Rad, Ramin, Huttlin, Edward L., Gygi, Steven P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4515955/
https://www.ncbi.nlm.nih.gov/pubmed/26076430
http://dx.doi.org/10.1038/nbt.3267
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author Chick, Joel M.
Kolippakkam, Deepak
Nusinow, David P.
Zhai, Bo
Rad, Ramin
Huttlin, Edward L.
Gygi, Steven P.
author_facet Chick, Joel M.
Kolippakkam, Deepak
Nusinow, David P.
Zhai, Bo
Rad, Ramin
Huttlin, Edward L.
Gygi, Steven P.
author_sort Chick, Joel M.
collection PubMed
description Fewer than half of all tandem mass spectrometry (MS/MS) spectra acquired in shotgun proteomics experiments are typically matched to a peptide with high confidence. Here we determine the identity of unassigned peptides using an ultra-tolerant Sequest database search that allows peptide matching even with modifications of unknown masses up to ±500 Da. In a proteome-wide dataset on HEK293 cells (9,513 proteins and 396,736 peptides), this approach matched an additional 184,000 modified peptides, which were linked to biological and chemical modifications representing 523 distinct mass bins, including phosphorylation, glycosylation, and methylation. We localized all unknown modification masses to specific regions within a peptide. Known modifications were assigned to the correct amino acids with frequencies often >90%. We conclude that at least one third of unassigned spectra arise from peptides with substoichiometric modifications.
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spelling pubmed-45159552016-01-01 An ultra-tolerant database search reveals that a myriad of modified peptides contributes to unassigned spectra in shotgun proteomics Chick, Joel M. Kolippakkam, Deepak Nusinow, David P. Zhai, Bo Rad, Ramin Huttlin, Edward L. Gygi, Steven P. Nat Biotechnol Article Fewer than half of all tandem mass spectrometry (MS/MS) spectra acquired in shotgun proteomics experiments are typically matched to a peptide with high confidence. Here we determine the identity of unassigned peptides using an ultra-tolerant Sequest database search that allows peptide matching even with modifications of unknown masses up to ±500 Da. In a proteome-wide dataset on HEK293 cells (9,513 proteins and 396,736 peptides), this approach matched an additional 184,000 modified peptides, which were linked to biological and chemical modifications representing 523 distinct mass bins, including phosphorylation, glycosylation, and methylation. We localized all unknown modification masses to specific regions within a peptide. Known modifications were assigned to the correct amino acids with frequencies often >90%. We conclude that at least one third of unassigned spectra arise from peptides with substoichiometric modifications. 2015-06-15 2015-07 /pmc/articles/PMC4515955/ /pubmed/26076430 http://dx.doi.org/10.1038/nbt.3267 Text en http://www.nature.com/authors/editorial_policies/license.html#terms Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms
spellingShingle Article
Chick, Joel M.
Kolippakkam, Deepak
Nusinow, David P.
Zhai, Bo
Rad, Ramin
Huttlin, Edward L.
Gygi, Steven P.
An ultra-tolerant database search reveals that a myriad of modified peptides contributes to unassigned spectra in shotgun proteomics
title An ultra-tolerant database search reveals that a myriad of modified peptides contributes to unassigned spectra in shotgun proteomics
title_full An ultra-tolerant database search reveals that a myriad of modified peptides contributes to unassigned spectra in shotgun proteomics
title_fullStr An ultra-tolerant database search reveals that a myriad of modified peptides contributes to unassigned spectra in shotgun proteomics
title_full_unstemmed An ultra-tolerant database search reveals that a myriad of modified peptides contributes to unassigned spectra in shotgun proteomics
title_short An ultra-tolerant database search reveals that a myriad of modified peptides contributes to unassigned spectra in shotgun proteomics
title_sort ultra-tolerant database search reveals that a myriad of modified peptides contributes to unassigned spectra in shotgun proteomics
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4515955/
https://www.ncbi.nlm.nih.gov/pubmed/26076430
http://dx.doi.org/10.1038/nbt.3267
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