A new liquid chromatography/tandem mass spectrometric approach for the identification of class I major histocompatibility complex associated peptides that eliminates the need for bioassays.
Cell-surface class I major histocompatibility complex (MHC) molecules present processed self- and nonself-peptides to thymus-derived (T) lymphocytes, allowing the intracellular compartment of cells to be sampled in order to detect infection. Since the class I MHC-peptide complex plays a critical role in cell-mediated immunity, it is important to obtain sequence information on the MHC-associated peptides unique to infected cells as a prelude to the development of vaccines. Here, we outline and test an alternative strategy for identifying the proteins that are processed through the MHC pathway. This new strategy eliminates the necessity of developing and maintaining cytotoxic T lymphocyte (CTL) lines for peptide identification. In this new approach genome sequences from the infecting agent are scanned for stretches of amino acids that match a particular MHC binding motif. Molecular masses from these putative MHC-binding peptide sequences are calculated and compared to those found for peptides isolated from pathogen-infected host cells using liquid chromatography/mass spectrometry (LC/MS). Peptides with masses matching those in the database are then analyzed by tandem mass spectrometry (MS/MS) to determine their identity. Using this approach we were able to confirm the processing and presentation of two Trypanosoma cruzi proteins by the MHC class I pathway. These data suggest that a rigorous approach employing two-dimensional separations in conjunction with MS/MS and bioinformatics is a feasible means of identifying pathogen gene products of immunological interest when a CTL assay is unavailable or unsuccessful.
Brockman
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AH
Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA.
Orlando
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R
Tarleton
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RL
D000818:Animals; D014355:Chagas Disease / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D017022:Flow Injection Analysis; D005805:Genes, MHC Class I / Q000235:genetics*; D013058:Mass Spectrometry; D051379:Mice; D008970:Molecular Weight; D009206:Myocardium / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D014349:Trypanosoma cruzi
D010455:Peptides