Premium
Identification of human myocardial proteins separated by two‐dimensional electrophoresis with matrix‐assisted laser desorption/ionization mass spectrometry
Author(s) -
Thiede Bernd,
Otto Albrecht,
ZimnyArndt Ursula,
Müller EvaChristina,
Jungblut Peter
Publication year - 1996
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150170330
Subject(s) - chemistry , chromatography , mass spectrometry , bottom up proteomics , protein mass spectrometry , sample preparation in mass spectrometry , peptide mass fingerprinting , matrix assisted laser desorption/ionization , isobaric labeling , methionine , cysteine , peptide , trypsin , biochemistry , proteomics , amino acid , tandem mass spectrometry , electrospray ionization , desorption , enzyme , organic chemistry , adsorption , gene
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 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. 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.