Journal
ANALYTICAL CHEMISTRY
Volume 79, Issue 21, Pages 7984-7991Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac070553t
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Funding
- NIDA NIH HHS [P30 DA018310] Funding Source: Medline
- NIGMS NIH HHS [5 T32 GM070421, R01 GM059826, R01 GM067193, R01 GM067193-04, T32 GM070421, GM59826] Funding Source: Medline
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Proteomics has grown significantly with the aid of new technologies that consistently are becoming more streamlined. While processing of proteins from a whole cell lysate is typically done in a bottom-up fashion utilizing MS/MS of peptides from enzymatically digested proteins, top-down proteomics is becoming. a viable alternative that until recently has been limited largely to offline analysis by tandem mass spectrometry. Here we describe a method for high-resolution tandem mass spectrometery of intact proteins on a chromatographic time scale. In a single liquid chromatography-tandem mass spectrometry (LC-MS/MS) run, we have identified 22 yeast proteins with molecular weights from 14 to 35 kDa. Using anion exchange chromatography to fractionate a whole cell lysate before online LC-MS/MS, we have detected 231 metabolically labeled (N-14/N-15) protein pairs from Saccharomyces cerevisiae. Thirty-nine additional proteins were identified and characterized from LC-MS/MS of selected anion exchange fractions. Automated localization of multiple acetylations on Histone H4 was also accomplished on an LC time scale from a complex protein mixture. To our knowledge, this is the first demonstration of top-down proteomics (i.e., many identifications) on linear ion trap Fourier transform (LTQ FT) systems using high-resolution MS/MS data obtained on a chromatographic time scale.
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