Tandem mass spectrometry (MS/MS) allows for the rapid identification of many types of post-translational modifications (PTMs), especially those that can be detected by a diagnostic mass shift in one or more peptide fragment ions (for example, phosphorylation). But some PTMs (for example, SUMOs and other ubiquitin-like modifiers) themselves produce multiple fragment ions; combined with fragments from the modified target peptide, a complex overlapping fragmentation pattern is thus generated, which is uninterpretable by standard peptide sequencing software. Here we introduce SUMmOn, an automated pattern recognition tool that detects diagnostic PTM fragment ion series within complex MS/MS spectra, to identify modified peptides and modification sites within these peptides. Using SUMmOn, we demonstrate for the first time that human SUMO-1 multimerizes in vitro primarily via three N-terminal lysines, Lys7, Lys16 and Lys17. Notably, our method is theoretically applicable to any type of modification or chemical moiety generating a unique fragment ion pattern.
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We thank A.-C. Gingras, J. Eng, O. Vitek, X. Li, A. Nesvizhskii and N. Zhang for technical advice and critical reading of the manuscript. This project was funded in part by federal funds from the US National Heart, Lung and Blood Institute, National Institutes of Health, under contract number N01-HV-28179. X.D.Z. and M.J.M. were supported by a grant from the National Institutes of Health (GM060980). B.R. holds the Canada Research Chair (Tier 2) in Proteomics and Molecular Medicine, with support from the Canada Foundation for Innovation, the Ontario Innovation Trust, and the McLaughlin Centre for Molecular Medicine.
Screenshot of the introductory SUMmOn user interface. (PDF 456 kb)
SUMmOn identifies a known RanGAP1 conjugation site modified with SUMO-2 and a known RanGAP1 conjugation site modified with SUMO-3. (PDF 1788 kb)
SUMmOn identifies a known SUMO-2 conjugation site within SUMO-2. (PDF 320 kb)
SUMmOn identifies a known RanGAP1 conjugation site modified with Smt3. (PDF 1018 kb)
SUMmOn identifies SUMO conjugation sites in RanGAP1 and SUMO-1. (PDF 996 kb)
Identification of SUMO-1 multimerization sites. (PDF 540 kb)
Data used for modification site identification in this work. (PDF 93 kb)
SUMO sequence comparison. (PDF 218 kb)
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Nature Chemical Biology (2017)
Virologica Sinica (2017)
Nature Protocols (2016)
Nature Protocols (2015)
Nature Communications (2014)