IDENTIFICATION OF MYOCARDIAL PROTEINS FROM 2-DIMENSIONAL GELS BY PEPTIDE MASS FINGERPRINTING

Two-dimensional gels offer a powerful method for separating complex pr otein mixtures, but subsequent methods for analysing individual compon ents, such as protein sequencing and Western immunoblotting, are labor ious and slow. The identification of proteins can be accelerated by us ing a combination of protease digestion and matrix assisted laser deso rption-mass spectrometry (MALDI-MS). The peptide mass spectrum of a pr otein represents a unique fingerprint determined by the amino acid seq uence and the cleavage properties of the protease. Software has been d eveloped so that peptide masses can be used to search a mass-based pep tide database generated from established protein sequence databases. A list of the closest matching proteins is produced to allow identifica tion of the sample. The strategy was applied to 52 protein spots from human myocardial tissue separated by two-dimensional electrophoresis ( 2-DE) gels and analysed blind. Conditions for optimal trypsin digestio n of proteins electroblotted onto polyvinylidene difluoride (PVDF) mem branes are described. Mass data were generated from both Coomassie Bri lliant Blue and sulforhodamine B-stained proteins, though the former r equired destaining prior to digestion. Alkylation of cysteine and oxid ation of methionine were significant modifications that influenced the successful identification of a protein spot. Examples are presented t o illustrate the advantages and disadvantages of this approach.