IDENTIFICATION OF THE COMPONENTS OF SIMPLE PROTEIN MIXTURES BY HIGH-ACCURACY PEPTIDE MASS MAPPING AND DATABASE SEARCHING

Peptide mass mapping by matrix-assisted laser desorption/ionization (M ALDI) followed by database searching with the set of measured peptide masses is now a powerful method for the identification of pure protein s, Protein mixtures-such as frequently occur due to comigration in pol yacrylamide gel bands-have hitherto required protein sequencing, Here we demonstrate that such protein bands can also be analyzed by peptide mass mapping alone. Database searching with the complete list of pept ide masses determined by delayed-extraction MALDI mass spectrometry wi th a mass error of less than 30 ppm retrieves the most prominent prote in in a mixture. In a second step, the protein identity is further con firmed by matching as many of the measured peptide masses as possible to the retrieved amino acid sequence, Peptide masses remaining after t his ''second pass search'' are searched again to identify the next com ponent in the protein mixture. This iterative process is repeated unti l all major ion signals are accounted for. Protein mixtures consisting of two or more individual components in a single gel band can be anal yzed, further increasing the general applicability of MALDI peptide ma pping for protein identification.