Identification of MHC-Associated Peptides and Phosphopeptides in Various Cell Lines Utilizing Tandem Mass Spectrometry

All nucleated cells use Major Histocompatibility Complex (MHC) class I molecules to communicate their intracellular status to the immune system. The cytosolic proteasome in the cytosol degrades viral and self-derived proteins that do not reach their functional states(mutated), do not fold correctly or no longer needed by the cell. After degraded peptides are presented on the MHC molecules to the cell surface, activated cytotoxic T-cells (CTLs) interact with MHC class I complexes and recognize antigenic peptides.
One of the most common post-translational modifications (PTMs) is phosphorylation, essential in cellular signaling, including protein synthesis, cell division, and cell growth. Enzymes known as kinases and phosphatases facilitate phosphorylation and de-phosphorylation events, respectively. Disrupting or mutating the regulatory mechanisms of kinases and/or phosphatases leads to abnormal activation or deactivation of proteins, which can cause cancer and other illnesses. Therefore, in cancer, dysregulated cellular signaling pathways can lead to the presentation of peptides with aberrant phosphorylation. CTLs can recognize phosphopeptides associated with MHC class I molecules as antigenic and initiate a significant immune response against the malignant cells. Therefore, one of the leading targets for T-cell recognition of diseased cells is HLA associated peptides derived from the processing and presentation of mutated proteins.
The long-term goal of this investigation is to evaluate the T cell response triggered by tumor-specific phosphopeptide candidates. With the advancement in immunotherapy, we believe that our work can impact the potential eradication of cancer by incorporating candidate tumor-specific phosphopeptides into peptide-based vaccines In this dissertation, chapter one, our current approach for identifying MHC-associated phosphopeptides by using enhanced sample preparation protocols, complementary IMAC enrichment techniques (𝐹𝑒3+-NTA), and high-resolution mass spectrometry has been summarized. In
chapter two, human chronic lymphocytic leukemia (CLL) samples have been subjected to the phosphopeptide enrichment technique leading to the identification of 94 HLA associated phosphopeptides. The confirmed phosphopeptides were analyzed in terms of their presence in varying types of cancer previously analyzed in the Hunt lab. In chapter three-part one, human breast cancer cell lines grown in immunocompromised mice have been subjected to phosphopeptide enrichment technique leading to the identification of 163 HLA-associated phosphopeptides. In chapter three-part two, liver tissue and melanoma cell line derived from the C57BL/6 mouse strain have been immunopurified using allele specific antibodies (Y3 and B22-249) and pan-H2 antibody (M1). Our results show that M1-based MHC purification yields peptides typical of both individual MHC genes, while the widely used H-2Kb (Y3) and H-2Db (B22-249) allele-specific antibodies yielded peptides typical of only their alleles. In chapter four, the epidermoid carcinoma cell line's (A431) two genes (ALPP and/or ALPPL2) were knocked out using CRISPR technology. Then MHC class I associated phosphorylated peptides isolated from the cell surface of A431 samples were analyzed using a high-resolution mass spectrometry instrument. The number of peptides between samples agrees with our hypothesis that upregulation of ALPP or ALPPL2 prevents the presentation of phosphopeptides through the MHC class I pathway in many cancers