期刊
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
卷 378, 期 4, 页码 1102-1111出版社
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-003-2159-8
关键词
affinity-proteomics; protein identification; biological material; mass spectrometric epitope mapping; immuno-affinity isolation; partial proteolytic degradation; matrix assisted laser desorption-ionisation; (MALDI) mass spectrometry; electrospray-ionisation (ESI) mass spectrometry; database analysis
We describe here a new approach for the identification of affinity-bound proteins by proteolytic generation and mass spectrometric analysis of their antibody bound epitope peptides (epitope excision). The cardiac muscle protein troponin T was chosen as a protein antigen because of its diagnostic importance in myocardial infarct, and its previously characterised epitope structure. Two monoclonal antibodies (IgG1-1B10 and IgG1-11.7) raised against intact human troponin T were found to be completely cross reactive with bovine heart troponin T. A combination of immuno-affinity isolation, partial proteolytic degradation (epitope excision), mass spectrometric peptide mapping, and database analysis was used for the direct identification of Tn T from bovine heart cell lysate. Selective binding of the protein was achieved by addition of bovine heart cell lysate to the Sepharose-immobilised monoclonal antibodies, followed by removal of supernatant material containing unbound protein. While still bound to the affinity matrix the protein was partially degraded thereby generating a set of affinity-bound, overlapping peptide fragments comprising the epitope. Following dissociation from the antibody the epitope peptides were analysed by matrix assisted laser desorption-ionisation (MALDI) and electrospray-ionisation (ESI) mass spectrometry. The peptide masses identified by mass spectrometry were used to perform an automated database search, combined with a search for a common 'epitope motif'. This procedure resulted in the unequivocal identification of the protein from biological material with only a minimum number of peptide masses, and requiring only limited mass-determination accuracy. The dramatic increase of selectivity for identification of the protein by combining the antigen-antibody specificity with the redundancy of peptide sequences renders this 'affinity-proteomics' approach a powerful tool for mass spectrometric identification of proteins from biological material.
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