High throughput protein characterization by automated reverse‐phase chromatography/electrospray tandem mass spectrometry

We describe an integrated workstation for the automated, high‐throughput, and conclusive identification of proteins by reverse‐phase chromatography electrospray ionization tandem mass spectrometry. The instrumentation consists of a refrigerated autosampler, a submicrobore reverse‐phase liquid chromatograph, and an electrospray triple quadrupole mass spectrometer. For protein identification, enzymatic digests of either homogeneous polypeptides or simple protein mixtures were generated and loaded into the autosampler. Samples were sequentially injected every 32 min. Ions of eluting peptides were automatically selected by the mass spectrometer and subjected to collision‐induced dissociation. Following 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 databases. Protein identification was established by SEQUEST_SUMMARY a program that combines the SEQUEST scores of peptides originating from the same protein and ranks the cumulative results in a short summary.

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