文摘
For improved detection of diverse posttranslational modifications (PTMs), direct fragmentation of protein ions bytop down mass spectrometry holds promise but has yetto be achieved on a large scale. Using lysate from Saccharomyces cerevisiae, 117 gene products were identified with 100% sequence coverage revealing 26 acetylations, 1 N-terminal dimethylation, 1 phosphorylation, 18duplicate genes, and 44 proteolytic fragments. The platform for this study combined continuous-elution gelelectrophoresis, reversed-phase liquid chromatography,automated nanospray coupled with a quadrupole-FThybrid mass spectrometer, and a new search engine forquerying a custom database. The proteins identifiedrequired no manual validation, ranged from 5 to 39 kDa,had codon biases from 0.93 to 0.083, and were primarilyassociated with glycolysis and protein synthesis. Illustrations of gene-specific identifications, PTM detection andsubsequent PTM localization (using either electron capture dissociation or known PTM data stored in a database)show how larger scale proteome projects incorporating topdown may proceed in the future using commercial Q-FTinstruments.