Gas-Phase Enrichment of Multiply Charged Peptide Ions by Differential Ion Mobility Extend the Comprehensiveness of SUMO Proteome Analyses
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The small ubiquitin-like modifier (SUMO) is a member of the family of ubiquitin-like modifiers (UBLs) and is involved in important cellular processes, including DNA damage response, meiosis and cellular trafficking. The large-scale identification of SUMO peptides in a site-specific manner is challenging not only because of the low abundance and dynamic nature of this modification, but also due to the branched structure of the corresponding peptides that further complicate their identification using conventional search engines. Here, we exploited the unusual structure of SUMO peptides to facilitate their separation by high-field asymmetric waveform ion mobility spectrometry (FAIMS) and increase the coverage of SUMO proteome analysis. Upon trypsin digestion, branched peptides contain a SUMO remnant side chain and predominantly form triply protonated ions that facilitate their gas-phase separation using FAIMS. We evaluated the mobility characteristics of synthetic SUMO peptides and further demonstrated the application of FAIMS to profile the changes in protein SUMOylation of HEK293 cells following heat shock, a condition known to affect this modification. FAIMS typically provided a 10-fold improvement of detection limit of SUMO peptides, and enabled a 36% increase in SUMO proteome coverage compared to the same LC-MS/MS analyses performed without FAIMS.
KeywordsHigh-field asymmetric waveform ion mobility spectrometry (FAIMS) Proteomics SUMOylation Heat shock
The authors thank Jean-Jacques Dunyach, Michael Belford and Satendra Prasad (Thermo Fisher Scientific) for valuable help and assistance with the FAIMS interface. The Institute for Research in Immunology and Cancer (IRIC) receives infrastructure support from IRICoR, the Canadian Foundation for Innovation, and the Fonds de Recherche du Québec-Santé (FRQS). IRIC proteomics facility is a Genomics Technology platform funded in part by the Canadian Government through Genome Canada.
This work was carried out with financial support from the Natural Sciences and Engineering Research Council (NSERC 311598) and the Genomic Applications Partnership Program (GAPP) of Genome Canada. The Institute for Research in Immunology and Cancer (IRIC) receives infrastructure support from IRICoR, the Canadian Foundation for Innovation, and the Fonds de Recherche du Québec - Santé (FRQS). IRIC proteomics facility is a Genomics Technology platform funded in part by the Canadian Government through Genome Canada.
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