Journal
ANALYTICAL CHEMISTRY
Volume 78, Issue 3, Pages 686-694Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac050993p
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Funding
- NIGMS NIH HHS [GM 067193, GM 59826] Funding Source: Medline
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The extension of quantitation methods for small peptides to ions above 5 kDa, and eventually to global quantitative proteomics of intact proteins, will require extensive refinement of current analytical approaches. Here we evaluate postgrowth Cys-labeling and N-14/N-15 metabolic labeling strategies for determination of relative protein expression levels and their posttranslational modifications using top-down mass spectrometry (MS). We show that intact proteins that are differentially alkylated with acrylamide (+71 Da) versus iodoacetamide (+57 Da) have substantial chromatographic shifts during reversed-phase liquid chromatography separation (particularly in peak tails), indicating a requirement for stable isotopes in alkylation tags for top-down MS. In the N-14/N-15 metabolic labeling strategy, we achieve 98% N-15 incorporation in yeast grown 10 generations under aerobic conditions and determine 50 expression ratios using Fourier transform ion cyclotron resonance MS in comparing these cells to anaerobically grown control (N-14) cells. We devise quantitative methods for top-down analyses, including a correction factor for accurate protein ratio determination based upon the signal-to-noise ratio. Using a database of 200 yeast protein forms identified previously by top-down MS, we verify the intact mass tag concept for protein identification without tandem MS. Overall, we find that top-down MS promises work flows capable of large-scale proteome profiling using stable isotope labeling and the determination of > 5 protein ratios per spectrum.
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