Top-Down Approaches for Measuring Expression Ratios of Intact Yeast Proteins Using Fourier Transform Mass Spectrometry

• Published in: Analytical chemistry (Washington) Vol. 78; no. 3; pp. 686 - 694
• Main Authors: Du, Yi, Parks, Bryan A, Sohn, Seyoung, Kwast, Kurt E, Kelleher, Neil L
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.02.2006
• Subjects: Analytical chemistry; Chemistry; Chromatographic methods and physical methods associated with chromatography; Exact sciences and technology; Feasibility Studies; Fourier Analysis; Fourier transforms; Gene expression; Mass spectrometry; Molecular Weight; Other chromatographic methods; Proteomics - methods; Saccharomyces cerevisiae - chemistry; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae Proteins - analysis; Sensitivity and Specificity; Spectrometric and optical methods; Spectrometry, Mass, Electrospray Ionization - instrumentation; Spectrometry, Mass, Electrospray Ionization - methods; Yeast; Fourier transform spectroscopy; Peptides; Liquid chromatography; Correction factor; Alkylation; Protein; Acrylamide; Reversed phase chromatography; Mass spectrometry MS/MS; Database; Ion cyclotron resonance; Mass spectrometry; Quantitative analysis; Signal to noise ratio
• DOI: 10.1021/ac050993p
• ISSN: 0003-2700

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 14N/15N 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 14N/15N metabolic labeling strategy, we achieve 98% 15N 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 (14N) 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.