Improved methods for identification of MHC class I-associated peptides
Mahoney, Keira, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Hunt, Donald, AS-Chemistry, University of Virginia
This dissertation presents three projects, each focusing on the identification of peptides presented to the immune system through the major histocompatibility complex class I pathway. This pathway is responsible for digesting intracellular proteins into peptides and presenting these peptides to circulating CD8+ T cells, providing all nucleated cells with a mechanism to communicate their health to the immune system. Often, in transformed or diseased cells, dysregulated cellular signaling leads to altered protein expression and/or upregulation in post-translational modifications. In particular, increased phosphorylation is a hallmark of cancer and translates to an increase in phosphopeptide presentation via the MHC class I processing pathway. In many cases, we have observed the same phosphorylated peptides presented across different cancerous tissues. Thus, the first project explores the reason for this, and presents early evidence that the same phosphorylated peptides may be presented by other chronic diseases. This information would allow for immunotherapeutics targeting phosphopeptide antigens to be used for a variety of different diseases.
In order to detect phosphopeptides by mass spectrometry, it is necessary to perform enrichment for phosphorylation prior to MS analysis. Unfortunately, the enrichment procedure used in the Hunt laboratory is extremely laborious and commonly involves issues that prevent the final analysis of phosphopeptides. In the second project presented here, we significantly improved our phosphopeptide enrichment protocol. These improvements maintain high sensitivity and specificity while reducing the length and difficulty of the process. Another issue regarding phosphopeptide detection and enrichment is the losses of hydrophilic peptides during the steps used to remove contaminants from the samples prior to analysis. Thus, the third project explores a hydrophilic interaction-based protocol that I developed to remove contaminants from our samples has since been modified and adopted to a variety of other applications. Prior to the improvements described within, the most phosphopeptides identified in a single sample was 234 using two enrichments and 1 billion cells. After these advances, we identified 522 phosphorylated peptides using 100 million cells, thus demonstrating the utility of the protocol improvements. Together, this work presents significant advances in immunoproteomics, particularly phosphopeptide analysis.
PHD (Doctor of Philosophy)
immunopeptidomics, proteomics, human leukocyte antigen (HLA), mass spectrometry
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