Determination of Relative Expression Levels of HLA Class-I Proteins in Peripheral Blood Lymphocytes Using Immunoprecipitationin Combination with Selected Ion Monitoring Mass Spectrometry

摘要

Human leukocyte antigens (HLA) is a family of molecules essential for immune function and with diverse clinical implications in infectious disease, autoimmunity, transplantation, cancer and pregnancy. Expression level of the human leukocyte antigens (HLAs) is known to influence pathological outcomes. Recent findings show a higher level of HLA-C expression is protective during HIV infection in multiple ethnicities and correlates with greater frequencies of both HIV-specific T-cell responses and viral escape mutations [1]. Classical HLA class-I loci relative expression levels are of particular interest on HIV-infected cells. Although expression level has important consequences for HLA function, relative levels of proteins expressed from the HLA-A, -B, -C, and -E loci are not well established. Precise quantitation of the HLA-A, -B, -C, and -E protein expression levels on normal primary cells as well as on HIV-infected cells would be valuable. The high homology (>80%) of the HLA variants of interest made it difficult to develop an antibody-based approach with antibodies specific for each protein with comparable affinities. Mass spectrometry, however, has the ability to directly detect and quantify highly homologous proteins if they have unique peptide sequences that distinguish them. Here, we describe the determination of the relative expression levels of HLA-A, HLA-B, and HLA-C for the specific haplotype A~*02:01, B~*44:02, C~*05:01, and HLA-E proteins from normal primary cells using selected ion monitoring mass spectrometry targeting their unique peptides in combination with stable isotope-labeled synthetic peptides for their absolute quantitation after total HLA immunoprecipitation.