Progress Toward Peptide-Based Cancer Immunotherapies: Identification of Class I MHC-Associated Phosphorylated Peptides by Mass Spectrometry

Mass spectrometry has risen to the forefront of analytical techniques utilized to investigate biological samples due to its speed and sensitivity, which generates data that allows the unambiguous identification of post-translational modifications (PTMs) on proteins and peptides. Chief among these PTMs is phosphorylation, which controls many signaling pathways; when these pathways are dysregulated, malignant transformation often occurs. This dissertation describes the study of immunologically relevant samples; the work was performed using mass spectrometry with collisionally activated dissociation (CAD) and electron transfer dissociation (ETD). The major histocompatibility complex (MHC) I pathway generates endogenous peptides that are displayed on the cell surface and that communicate the health of a given cell to the immune system through signaling. This presentation system provides a way for the regulation of abnormal cells, like tumors, which are often marked as different from healthy cells through the peptides found on their surfaces. In the case of cancer, this distinction may arise from the differential display of phosphopeptides, as it is known that signaling pathways are often dysregulated in cancer, resulting in aberrant phosphorylation. We hypothesize that these differentially phosphorylated peptides displayed by MHC I molecules on the cell surface may act as a warning of developing malignancies. By identifying the phosphopeptides unique to cancer cells, it may be possible to develop peptide-based immunotherapies and biomarkers that can be used for the treatment and earlier diagnosis of cancer. Through the application of enrichment and separation techniques used in combination with mass spectrometry, we were able to identify 231 unique phosphopeptides displayed on a variety of melanoma and control cell lines. A preliminary analysis shows that several of the source proteins are known to be involved with malignant transformation, making those peptides targets for immunotherapeutic development. The final section of the dissertation presents a brief review of developing peptide-based immunotherapies. These treatments are exploring novel uses for peptides which extend their use beyond traditional vaccine therapy. Also discussed in this section is the possibility for phosphopeptides to be employed as a diagnostic tool, with this potential preliminarily demonstrated by our research into leukemia.