Joint work with Andrew J. Link, David A. Schieltz, Jimmy K. Eng, and Edwin Carmack.
Genomic and EST sequencing projects are providing a sequence infrastructure that is changing how protein biochemistry is practiced. Data produced by tandem mass spectrometers provides independent pieces of data about the components of a mixture. By applying tandem mass spectrometry to the analysis of protein mixtures the identities of the components can be determined by the appropriate application of software in conjunction with sequence databases. The approach we have developed uses proteolytic digestion of the protein complex followed by separation of the resulting mixture of peptides using microcolumn liquid chromatography interfaced to a tandem mass spectrometer. Data-dependent operation of the tandem mass spectra allows the highly efficient acquisition of tandem mass spectra. As peptide mixtures become more complex, highly resolving separation techniques are required to separate the peptide components for tandem mass spectrometry. We have examined several strategies to separate complex peptide mixtures using microcolumn LC/LC and SPE/CE in conjunction with tandem mass spectrometry. This approach has been applied to the analysis of large protein complexes and to total cellular proteins.