Abstract
Mass spectrometry is an invaluable tool in the study of proteomics. Through the use of mass spectrometry, protein binding partners have been identified, post translational modifications (PTMs) have been site-mapped, and the immunopeptidome has been characterized. The invention of electron transfer dissociation (ETD) greatly facilitated the ability to use mass spectrometry to site-map PTMs. This dissertation discusses four studies that apply and extend the use of mass spectrometry to better understand disease processes, adverse drug reactions, and immunology.
While ETD has enhanced the capability of site-mapping labile PTMs, it does have inherent limitations. ETD requires a charge state of at least +3 or higher to achieve optimal fragmentation, and in areas of charge deficiency sequence ions are not prroduced. This can cause difficulty when attempting to site map O-GlcNAc modifications because they typically occur in poly S/T regions within a peptide, which lack sufficient charge. To overcome this limitation, we have developed a method of adding charge to the O-GlcNAc moiety via the Staudinger reaction to effectively increase charge and enhance fragmentation via ETD.
Lamin A is an intermediate filament nuclear protein, which primarily provides mechanical support and stability to the nucleus. Mutations in the LMNA gene, which encodes lamin A, can result in a number of diseases. In the case of Hutchinson-Gilford progeria syndrome (HGPS), a single point mutation causes a deletion of fifty amino acids and causes changes to nuclear shape. Within this 50 amino acid deletion, targets for PTMs are deleted, including both known targets for O-GlcNAcylation. This dissertation reports the identification of O-GlcNAcylation sites on WT lamin A and the HGPS related Δ35 deletion lamin A to better understand the role O-GlcNAcylation plays in HGPS.
The Major Histocompatibility Complex (MHC) is an essential part of the immune system, which displays peptide antigens to T cells to assess the health of individual cells. If the presented peptide is identified as foreign, or ‘non-self’ then an immune response is elicited. Understanding the MHC peptidome, peptides presented by MHC molecules, can provide information necessary to understanding mechanisms of disease as well as provide insight into the adverse effects of pharmaceutical drugs. This dissertation reports the characterization of the Chinese rhesus macaque’s MHC peptidome by mass spectrometry to provide information relative to the development of an HIV/SIV vaccine.
Finally, adverse drug reactions (ADR), which cause immune-mediated responses can be linked to a specific MHC allele. For example, abacavir, a nucleoside analog used in the treatment of HIV, exclusively causes an ADR in individuals expressing the HLA-B*57:01 allele. MHC peptides from untreated cells and from abacavir treated cells were analyzed by mass spectrometry to identify potential differences between them. An altered peptide repertoire is presented in the presence of abacavir, stimulating a T cell response. This is a novel mechanism of T cell stimulation, which can be applied to other drugs causing an ADR.
