Automated high through put analysis of peptides associated with MHC class II molecules
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文摘
T cells recognize antigens in the form of short peptide fragments bound to the products of major histocompatibility complex (MHC). MHC class I and class II molecules derive their peptides from intracellular protein breakdown in the cytosol and in lysosomal compartment, respectively. Thus, MHC molecules probe intracellular “garbage” to present the immune system with “fingerprints” of intruders - peptides derived from proteins foreign to the organism. T cells discriminate between endogenous and foreign antigenic peptides associated with MHC class I and class II. Sequence analysis of MHC associated peptides is a daunting task because of the extreme complexity of peptide mixtures which associate with any given MHC molecule. We have developed a high through put automated sequence analysis of peptides associated with MHC molecules using electrospray ionization tandem mass-spectrometry. Interpretation of tandem mass spectra was achieved by emplying a computer algorithm SEQUEST which uses protein and nucleotide databases to do correlation analysis of experimental and theoretical mass spectra generated from the database to obtain a peptide sequence. Using this approach an extensive database of over 100 endogenous peptides associated with murine MHC class II molecule I-Ab in B cells and macrophages was obtained. A substantial proportion of these peptides are derived from a subset of cytosolic proteins associated with cellular membranes or derived from cytosolic domains of transmembrane proteins. Subsequent functional experiments indicated expression of corresponding peptide/MHC class II complexes in vivo. These observations suggest a pathway(s) of class II presentation of some cytosolic proteins selectively imported into lysosomal compartment where peptide loading on MHC class II molecules takes place.