Top-Down Approaches for Measuring Expression Ratios of Intact Yeast Proteins Using Fourier Transform Mass Spectrometry
- 23 December 2005
- journal article
- research article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 78 (3), 686-694
- https://doi.org/10.1021/ac050993p
Abstract
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.Keywords
This publication has 34 references indexed in Scilit:
- Measurement of the Isotope Enrichment of Stable Isotope-Labeled Proteins Using High-Resolution Mass Spectra of PeptidesAnalytical Chemistry, 2005
- Molecular-Level Description of Proteins from Saccharomyces cerevisiae Using Quadrupole FT Hybrid Mass Spectrometry for Top Down ProteomicsAnalytical Chemistry, 2004
- Mass Spectrometric Interrogation of Thioester-Bound Intermediates in the Initial Stages of Epothilone BiosynthesisCell Chemical Biology, 2004
- Fourier-transform mass spectrometry for automated fragmentation and identification of 5-20 kDa proteins in mixturesElectrophoresis, 2002
- Genomic Analyses of Anaerobically Induced Genes in Saccharomyces cerevisiae : Functional Roles of Rox1 and Other Factors in Mediating the Anoxic ResponseJournal of Bacteriology, 2002
- Stored waveform inverse Fourier transform (SWIFT) ion excitation in trapped-ion mass spectometry: Theory and applicationsInternational Journal of Mass Spectrometry and Ion Processes, 1996
- ATP Synthase of Yeast MitochondriaPublished by Elsevier BV ,1996
- Application of linear prediction to Fourier transform ion cyclotron resonance signals for accurate relative ion abundance measurementsAnalytical Chemistry, 1992
- Initial relative ion abundances and relaxation times from apodized, segmented FT/ICR time domain signalsInternational Journal of Mass Spectrometry and Ion Processes, 1990
- Segmented fourier transform and its application to fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry: ion abundances and mass measurementsInternational Journal of Mass Spectrometry and Ion Processes, 1989