Identification of human myocardial proteins separated by two-dimensional electrophoresis with matrix-assisted laser desorption/ionization mass spectrometry.
Disease-associated proteins separated by two-dimensional electrophoresis (2-DE) are often in the femtomole range. Identification of 2-DE separated proteins by sequencing and amino acid analysis is limited to the lower picomole range. Identification down to the femtomole range can be achieved by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). We optimized the measurement by MALDI-MS for the analysis of proteolytic digests of 2-DE-separated proteins. The direct analysis of peptide mixtures can be used for rapid and sensitive protein identification. In some cases, more information about the protein can be obtained by separating the peptides by micro high-performance liquid chromatography (HPLC) before employing MALDI-MS analysis. More peptides are found than in the mixtures, 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. Furthermore, 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 oxidation and alkylation of cysteine by acrylamide into the mass search. By applying this procedure, 15 proteins were identified, among them two different putative phosphorylated forms of two proteins, a putative N-terminal blocking group and four dilated cardiomyopathy-associated proteins. The resulting approach for the identification may be used for large-scale investigations of 2-DE-separated proteins.
Thiede
B
B
Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany.
Otto
A
A
Zimny-Arndt
U
U
Müller
E C
EC
Jungblut
P
P
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000032:analysis; D003459:Crystallins / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D006801:Humans; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000032:analysis; D018996:Myelin P2 Protein / Q000032:analysis; D009206:Myocardium / Q000737:chemistry*; D018994:Myosin Light Chains / Q000032:analysis; D009363:Neoplasm Proteins; D010449:Peptide Mapping; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D025521:Tumor Suppressor Proteins
D002352:Carrier Proteins; D003459:Crystallins; C107608:FABP7 protein, human; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D009124:Muscle Proteins; D018996:Myelin P2 Protein; D018994:Myosin Light Chains; D009363:Neoplasm Proteins; D025521:Tumor Suppressor Proteins