HIGH-THROUGHPUT PROTEIN CHARACTERIZATION BY AUTOMATED REVERSE-PHASE CHROMATOGRAPHY ELECTROSPRAY TANDEM MASS-SPECTROMETRY

We describe an integrated workstation for the automated, high-throughp ut, and conclusive identification of proteins by reverse-phase chromat ography electrospray ionization tandem mass spectrometry, The instrume ntation consists of a refrigerated autosampler, a submicrobore reverse -phase liquid chromatograph, and an electrospray triple quadrupole mas s spectrometer. For protein identification, enzymatic digests of eithe r homogeneous polypeptides or simple protein mixtures were generated a nd loaded into the autosampler. Samples were sequentially injected eve ry 32 min, Ions of eluting peptides were automatically selected by the mass spectrometer and subjected to collision-induced dissociation. Fo llowing each run, the resulting tandem mass spectra were automatically analyzed by SEQUEST, a program that correlates uninterpreted peptide fragmentation patterns with amino acid sequences contained in database s, Protein identification was established by SEQUEST_SUMMARY a program that combines the SEQUEST scores of peptides originating from the sam e protein and ranks the cumulative results in a short summary. The wor kstation's performance was demonstrated by the unattended identificati on of 90 proteins from the yeast Saccharomyces cerevisiae, which were separated by high-resolution two-dimensional PAGE. The system was foun d to be very robust and identification was reliably and conclusively e stablished for proteins if quantities exceeding 1-5 pmol were applied to the gel. The level of automation, the throughput, and the reliabili ty of the results suggest that this system will be useful for the many projects that require the characterization of large numbers of protei ns.