IDENTIFICATION OF PROTEINS IN POLYACRYLAMIDE GELS BY MASS-SPECTROMETRIC PEPTIDE-MAPPING COMBINED WITH DATABASE SEARCH

Mass spectrometric peptide mapping of proteins separated by one-dimens ional sodium dodecyl sulphate polyacrylamide gel electrophoresis has b een investigated. The best results are obtained after blotting of the proteins onto polyvinylidene difluoride membranes followed by enzymati c digestion of the protein on the membrane. The peptide maps were inve stigated in terms of completeness and applicability for protein identi fication using a previously developed database search program as well as for the possibility for full characterization of covalent modificat ions in the proteins. The most complete peptide maps were obtained whe n the proteins were reduced and alkylated on the membrane prior to enz ymatic digestion followed by separation of the resulting mixture by hi gh performance liquid chromatography prior to mass spectrometric analy sis. Such peptide maps cover up to 98% of the sequence and consequentl y may allow complete characterization of post-translational modificati ons in proteins for which the amino acid sequence is known. The fastes t and most sensitive procedure to obtain peptide maps sufficient for p rotein identification was direct analysis of the extracted peptide mix ture by matrix-assisted laser desorption ionization (MALDI) mass spect rometry. The use of external and internal calibration of MALDI spectra for database searches is evaluated as well as the possibility of incl uding a post-calibration routine within the search program.