This is a revised proposal for the amino acid sequence analysis by mass spectrometry of antigens displayed on cell surfaces by Class I MHC (multiple histocompatibility [Sic ]) molecules for recognition by cytotoxic T lymphocytes (CTL) or killer T cells. At the time of our original submission of this proposal we had been successful in isolating, purifying, and sequencing an immunodominant H-2Kd-restricted NP147-155 (nucleoprotein) peptide isolated from virally transduced CT26NP cells and a Tap-dependent leader peptide recognized by alloreactive T cells specific for Class IB antigen using combinations of aminopeptidases, carboxypeptidases, and exopeptidases and matrix assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. While this approach has enabled us to develop efficient chromatographic methods for isolation of bioactive fractions and has established the detection limits (number of cells) required for sequencing, our interest is in identifying antigens specific to tumor or virally infected (including HIV) cells, that are displayed int the presence of a large number of self antigens that cannot be complete;y isolated chromatographically. Thus, our proposal was to employ tandem time of flight (TOF) mass spectrometry using a curved-field reflectron for simultaneous focussing of product sequence ions and a high performance quadrupole ion trap mass spectrometer (ITMS) using stored waveform inverse Fourier transform (SWIFT) techniques for unit mass selection, excitation, and collision induced dissociation (CID). In this revised proposal we address several concerns expressed by the reviewers, stressing the continued importance of off-line chromatography in combination with tandem instruments and detailing our methods for the development of a Salmonella -specific T cell assay. In addition, we describe our progress in reducing the chromatographic effort, in one case through development of a novel immunoprecipitation strategy for the isolation of Class IB antigen using a single HPLC fractionation. We also describe our recent improvements in performance (and commercialization) of the curved field reflectron mass spectrometer, as well as results in obtaining monoisotopic CID spectra using SWIFT techniques and pulsed heavy target gas injection in an ITMS. Finally, we describe two new projects that are now underway: the identification of MHC class I peptides associated with MMTV-infected mouse mammary tumor cells, and natural ligands recognized during T-cell selection in the thymus.
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