Measuring the Dynamics of the Proteome
- Department of Chemistry and Biochemistry and Institute of Cell and Molecular Biology, University of Texas, Austin, Texas 78712, USA
This extract was created in the absence of an abstract.
The modest goal of proteomics is to collect protein expression data to the same extent that one can now collect mRNA expression data with DNA microarrays. If proteomics lives up to its promise, we can expect to catalog thousands of different proteins from a sample of cells, then vary the cell growth conditions and see how the protein expression changes. Several technologies are vying to deliver on this promise, among them protein microarrays (MacBeath and Schreiber 2000). Currently, the most effective technique for cataloging the thousands of proteins in an average cell sample is mass spectrometry.
Studying the Proteome by Mass Spectrometry
The mass spectrometry proteomics experiments currently come in two general flavors: In one approach, proteins from a cell extract are first separated by two-dimensional (2D) gel electrophoresis. Then, proteins on the gel are identified by measuring their masses with MALDI-TOF mass spectrometry. The coups of this method include identifying many proteins from the yeast proteome (Shevchenko et al. 1996).
A second approach rids itself of the reliance on 2D electrophoresis and, therefore, promises to be technically simpler and more scaleable. In this approach (Fig. 1), proteins from a cell extract are first proteolytically digested into fragments (Hunt et al. 1986). The fragments are then partially purified by high-performance liquid chromatography and injected into an electrospray tandem mass spectrometer (MS/MS) or ion-trap mass spectrometer (LCQ). The mass spectrometer efficiently separates the peptide mixtures. In a continuous, automatic process, each peptide peak is in turn selected from the peptide mixture flowing into the mass spectrometer and sequenced by fragmenting the peptide in a collision cell and then measuring the masses of the peptide fragments.
The combination of HPLC and tandem mass spectrometry allows the identification of potentially thousands of proteins from complex samples. In this approach to proteomics, mass spectra are generated for proteolytic peptides …
