IDENTIFICATION OF HUMAN MYOCARDIAL PROTEINS SEPARATED BY 2-DIMENSIONAL ELECTROPHORESIS WITH MATRIX-ASSISTED LASER-DESORPTION IONIZATION MASS-SPECTROMETRY

Disease-associated proteins separated by two-dimensional electrophores is (2-DE) are often in the femtomole range. Identification of 2-DE sep arated proteins by sequencing and amino acid analysis is limited to th e lower picomole range. Identification down to the femtomole range can be achieved by matrix-assisted laser desorption ionization-mass spect rometry (MALDI-MS). We optimized the measurement by MALDI-MS for the a nalysis of proteolytic digests of 2-DE-separated proteins. The direct analysis of peptide mixtures can be used for rapid and sensitive prote in identification. In some cases, more information about the protein c an be obtained by separating the peptides by micro high-performance li quid chromatography (HPLC) before employing MALDI-MS analysis. More pe ptides are found than in the mixture, and comparison of HPLC patterns can reveal some differences to be post-translational modifications of proteins, even in the case of identical peptide mass fingerprints. Fur thermore, carboxy-terminal sequencing by on-target carboxypeptidase P digestion can be used to confirm the obtained result without the need for more material. The search program FRAGFIT was modified and renamed FRAGMOD to include the modifications of methionine and tryptophan oxi dation and alkylation of cysteine by acrylamide into the mass search, By applying this procedure, 15 proteins were identified, among them tw o different putative phosphorylated forms of two proteins, a putative N-terminal blocking group and four dilated cardiomyopathy-associated p roteins. The resulting approach for the identification may be used for large-scale investigations of 2-DE-separated proteins.