on 07-06-201601:36 AM - edited on 10-15-202105:42 AM by AnalyteGuru
Simone Sidoli, Rina Fujiwara and Benjamin A. Garcia Proteomics. 2016 May 19. doi: 10.1002/pmic.201500527. [Epub ahead of print] Wepresent the MS-based application of the innovative, although scarcely exploited, multiplexed data-independent acquisition (MSX-DIA) for the analysis of histone PTMs. Histones are golden standard for complexity in MS based proteomics, due to their large number of combinatorial modifications, leading to isobaric peptides after proteolytic digestion. DIA has, thus, gained popularity for the purpose as it allows for MS/MS-based quantification without upfront assay development. In this work, we evaluated the performance of traditional DIA versus MSX-DIA in terms of MS/MS spectra quality, instrument scan rate and quantification precision using histones from HeLa cells. We used an MS/MS isolation window of 10 and 6 m/z for DIA and MSX-DIA, respectively. Four MS/MS scans weremultiplexed for MSX-DIA. Despite MSX-DIA was programmed to perform two-fold more MS/MS events than traditional DIA, it acquired on average 5% more full MS scans, indicating even faster scan rate. Results highlighted an overall decrease of background ion signals usingMSX-DIA, and we illustrated specific examples where peptides of different precursor masses were co-fragmented by DIA but not MSX-DIA. Taken together, MSX-DIA proved thus to be a more favorable method for histone analysis in data independent mode.