"Affinity-proteomics": direct protein identification from biological material using mass spectrometric epitope mapping

Abstract

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.

Intact Transition Epitope Mapping – Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE)*

“…The same type of results, which match with reports from cross-reactivity-studies with diagnostic heart muscle troponin T antibodies (65), can be obtained with any epitope peptide amino acid sequence (Table I). Because of amino acid sequence similarities it is likely that the antibody of interest was able to recognize binding motifs on unrelated proteins, as long as the targeted amino acid sequence was surface exposed and the partial peptide assumed a somewhat similar three dimensional structure compared with that of the original epitope peptide, even when the respective antibody was not raised against the unrelated protein.…”

“…suitable for immune assays within a research project of a different species. Based on epitope peptide sequence similarities, the applicability of a precious antibody was securely broadened (8,65). Practicality of this approach is illustrated by NCBI BLAST searches using the here identified epitope peptide sequences without taxonomy restrictions.…”

Human monoclonal antibodies derived from a patient infected with 2009 pandemic influenza A virus broadly cross-neutralize group 1 influenza viruses

“…The epitopes recognized by the HuMAbs were analyzed by protease digestion, followed by mass spectrometry, as described previously [25]. Briefly, MAb-immobilized columns were prepared by adding each of the HuMAbs or C179 to HiTrap NHS-activated HP columns (GE Healthcare), followed by addition of recombinant HA protein (A/California/07/2009, 2 µg/ml, Protein Sciences).…”

Epitope Fishing in Digested Biological Samples

“…Our work is based on the same principle as "epitope extraction", which is a common method for identification of protein epitopes [11][12][13][14][15][16][17][18] . However, the way of applying it, is novel.…”