Identification of Class I and II MHC Peptides as Targets for Immunotherapy using Mass Spectrometry

Mass spectrometry is a powerful tool for detecting and identifying peptides and proteins. Tandem mass spectrometry (MS/MS) involves multiple steps of mass selection or analysis, which facilitates unambiguous assignment of peptide sequences and specific sites of modifications. Mass spectrometers have different resolutions and can be equipped with various fragmentation techniques. Mass spectrometric analysis has high sensitivity enabling analysis of attomole levels of peptides. The combination of mass spectrometry and chromatography techniques can advance the studies of innumerable biological systems including immunology. The first study focused on the identification of major histocompatibility complex (MHC) class I epitopes from Chlamydia pneumoniae (Cpn)-infected cells. The goal of this project is to identify Cpn-peptides in a complex mixture. An on-column N-terminal derivatization technique was used for sequencing of the class I peptides. The antigens found define multiple putative inclusion membrane proteins as targets of infection-primed CD8+ T cells. The second and third studies describe a phosphoproteomics analysis of class I and class II MHC peptides presented on cancer cells. Detection of phosphopeptides represents a significant analytical challenge since over 10,000 peptide species are displayed on the cell surface by MHC molecules and range in abundance from 1 to over 1000 copies per cell. Immobilized metalaffinity chromatography (IMAC) was utilized to isolate the phosphopeptides. Electron transfer dissociation (ETD) was performed for tandem mass spectrometric analysis since majority of the HLA-B7 and HLA-DR presenting peptides have high charge states and phosphopeptides are poorly fragmented under the conventional fragmentation technique collision activated dissociation (CAD). Our analysis of peptides presented by HLA-DR alleles detected and identified, for the first time, a list of phosphopeptides presented by class II molecules. Implications of these findings for the detection and prevention of cancer, especially leukemia and melanoma diagnostics and immunotherapeutics, will be discussed.