TY - JOUR
T1 - Molecular-level description of proteins from Saccharomyces cerevisiae using quadrupole FT hybrid mass spectrometry for top down proteomics
AU - Meng, Fanyu
AU - Du, Yi
AU - Miller, Leah M.
AU - Patrie, Steven M.
AU - Robinson, Dana E.
AU - Kelleher, Neil L.
PY - 2004/5/15
Y1 - 2004/5/15
N2 - 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.
AB - 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.
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U2 - 10.1021/ac0354903
DO - 10.1021/ac0354903
M3 - Article
C2 - 15144197
AN - SCOPUS:2442636680
SN - 0003-2700
VL - 76
SP - 2852
EP - 2858
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 10
ER -