Journal
JOURNAL OF PROTEOME RESEARCH
Volume 12, Issue 3, Pages 1520-1525Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr301130k
Keywords
electron transfer dissociation; ETD; HCD; EThcD; phosphorylation site localization; phosphoRS
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Funding
- European Union [262067]
- Netherlands Proteomics Centre,Netherlands Genomics Initiative
- Netherlands Organization for Scientific Research (NWO) [700.10.429]
- European Commission
- Austrian Science Fund via the Special Research Program Chromosome Dynamics [SFB-F3402]
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We recently introduced a novel scheme combining electron-transfer and higher-energy collision dissociation (termed EThcD), for improved peptide ion fragmentation and identification. We reasoned that phosphosite localization, one of the major hurdles in high throughput phosphoproteomics, could also highly benefit from the generation of such EThcD spectra. Here, we systematically assessed the impact on phosphosite localization utilizing EThcD in comparison to methods employing either ETD or HCD, respectively, using a defined synthetic phosphopeptide mixture and also using a larger data set of Ti4+-IMAC enriched phosphopeptides from a tryptic human cell line digest In combination with a modified version of phosphoRS, we observed that in the majority of cases EThcD generated richer and more confidently identified spectra, resulting in superior phosphosite localization scores. Our data demonstrates the distinctive potential of EThcD for PTM localization, also beyond protein phosphorylation.
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