RAPID MASS-SPECTROMETRIC PEPTIDE SEQUENCING AND MASS MATCHING FOR CHARACTERIZATION OF HUMAN-MELANOMA PROTEINS ISOLATED BY 2-DIMENSIONAL PAGE

We report a general mass spectrometric approach for the rapid identifi cation and characterization of proteins isolated by preparative two-di mensional polyacrylamide gel electrophoresis. This method possesses th e inherent power to detect and structurally characterize covalent modi fications. Absolute sensitivities of matrix-assisted laser desorption ionization and high-energy collision-induced dissociation tandem mass spectrometry are exploited to determine the mass and sequence of subpi comole sample quantities of tryptic peptides. These data permit mass m atching and sequence homology searching of computerized peptide mass a nd protein sequence data bases for known proteins and design of oligon ucleotide probes for cloning unknown proteins. We have identified 11 p roteins in lysates of human A375 melanoma cells, including: alpha-enol ase, cytokeratin, stathmin, protein disulfide isomerase, tropomyosin, Cu/Zn superoxide dismutase, nucleoside diphosphate kinase A, galaptin, and triosephosphate isomerase. We have characterized several posttran slational modifications and chemical modifications that may result fro m electrophoresis or subsequent sample processing steps. Detection of comigrating and covalently modified proteins illustrates the necessity of peptide sequencing and the advantages of tandem mass spectrometry to reliably and unambiguously establish the identity of each protein. This technology paves the way for studies of cell-type dependent gene expression and studies of targe suites of cellular proteins with unpre cedented speed and rigor to provide information complementary to the o ngoing Human Genome Project.