title: Molecular-level description of proteins from saccharomyces cerevisiae using quadrupole FT hybrid mass spectrometry for top down proteomics.
abstract: 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.
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
authors.forename: Fanyu
authors.initials: F
authors.lastname: Meng
authors.forename: Yi
authors.initials: Y
authors.lastname: Du
authors.forename: Leah M
authors.initials: LM
authors.lastname: Miller
authors.forename: Steven M
authors.initials: SM
authors.lastname: Patrie
authors.forename: Dana E
authors.initials: DE
authors.lastname: Robinson
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
mesh_terms: 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
chemical_list: D029701:Saccharomyces cerevisiae Proteins