Rapid Quantitative Measurements of Proteomes by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
The patterns of gene expression, post-translational modifications, protein/biomolecular interactions, and how these may be affected by changes in the environment, cannot be accurately predicted from DNA sequences. Approaches for proteome characterization are generally based upon mass spectrometric analysis of in-gel digested two dimensional polyacrylamide gel electrophoresis (2-D PAGE) separated proteins, allowing relatively rapid protein identification compared to conventional approaches. This technique, however, is constrained by the speed of the 2-D PAGE separations, the sensitivity limits intrinsic to staining necessary for protein visualization, the speed and sensitivity of subsequent mass spectrometric analyses for identification, and the limited ability for accurate quantitative measurements based on differences in spot intensity. We are presently developing alternative approaches for proteomics based upon the combination of fast capillary electrophoresis, or other suitable chromatographic separations, and the high mass accuracy and sensitivity obtainable with unique Fourier transform ion cyclotron resonance (FTICR) mass spectrometers available at our laboratory. Several approaches are presently being pursued; one based upon the analysis of intact proteins and the second upon approaches for global protein digestion and accurate peptide mass analysis. Quantitation of protein/peptide levels are based on using two or more stable-isotope labeled versions of proteomes which are combined to obtain precise quantitation of relative protein abundances. We describe the status of our efforts towards the development of a high-throughput proteomics capability and present initial results for application to several microorganisms and discuss our efforts for extending the developed capability to mammalian proteomes.
Bacterial proteins, cyclotrons, mass spectrometry, proteome
Smith, Richard D.; Pasa-Tolić, Ljiljana; Lipton, Mary S.; Jensen, Pamela K.; Anderson, Gordon A.; Shen, Yufeng; Conrads, Thomas P.; Udseth, Harold R.; Belov, Mikhail E.; Veenstra, Timothy D.; and et al, "Rapid Quantitative Measurements of Proteomes by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry" (2001). Pharmaceutical Sciences Faculty Publications. 508.