Molecular-level description of proteins from saccharomyces cerevisiae using quadrupole FT hybrid mass spectrometry for top down proteomics.
For improved detection of diverse posttranslational modifications (PTMs), direct fragmentation of protein ions by top down mass spectrometry holds promise but has yet to 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, 18 duplicate genes, and 44 proteolytic fragments. The platform for this study combined continuous-elution gel electrophoresis, reversed-phase liquid chromatography, automated nanospray coupled with a quadrupole-FT hybrid mass spectrometer, and a new search engine for querying a custom database. The proteins identified required no manual validation, ranged from 5 to 39 kDa, had codon biases from 0.93 to 0.083, and were primarily associated with glycolysis and protein synthesis. Illustrations of gene-specific identifications, PTM detection and subsequent PTM localization (using either electron capture dissociation or known PTM data stored in a database) show how larger scale proteome projects incorporating top down may proceed in the future using commercial Q-FT instruments.
Meng
Fanyu
F
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Du
Yi
Y
Miller
Leah M
LM
Patrie
Steven M
SM
Robinson
Dana E
DE
Kelleher
Neil L
NL
D000107:Acetylation; D001331:Automation; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel; D005823:Genetics; D006019:Glycolysis; D013058:Mass Spectrometry / Q000379:methods*; D008745:Methylation; D008970:Molecular Weight; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D029701:Saccharomyces cerevisiae Proteins