Rapid mass spectrometric peptide sequencing and mass matching for characterization of human melanoma proteins isolated by two-dimensional PAGE.
We report a general mass spectrometric approach for the rapid identification and characterization of proteins isolated by preparative two-dimensional polyacrylamide gel electrophoresis. This method possesses the inherent power to detect and structurally characterize covalent modifications. 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 subpicomole sample quantities of tryptic peptides. These data permit mass matching and sequence homology searching of computerized peptide mass and protein sequence data bases for known proteins and design of oligonucleotide probes for cloning unknown proteins. We have identified 11 proteins in lysates of human A375 melanoma cells, including: alpha-enolase, cytokeratin, stathmin, protein disulfide isomerase, tropomyosin, Cu/Zn superoxide dismutase, nucleoside diphosphate kinase A, galaptin, and triosephosphate isomerase. We have characterized several posttranslational modifications and chemical modifications that may result from 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 large suites of cellular proteins with unprecedented speed and rigor to provide information complementary to the ongoing Human Genome Project.
Clauser
K R
KR
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143, USA.
Hall
S C
SC
Smith
D M
DM
Webb
J W
JW
Andrews
L E
LE
Tran
H M
HM
Epstein
L B
LB
Burlingame
A L
AL
D000595:Amino Acid Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D016208:Databases, Factual; D004798:Enzymes / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D007525:Isoelectric Focusing; D013058:Mass Spectrometry / Q000379:methods; D008545:Melanoma / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000302:isolation & purification; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured
D004798:Enzymes; D009363:Neoplasm Proteins; D010455:Peptides; D011506:Proteins