Hunt, Donald F.
- BS, Chemistry, University of Massachusetts, Amherst, MA
- PhD, Organic Chemistry, University of Massachusetts, Amherst, MA
- Postdoc, Mass Spectrometry, MIT, Cambridge, MA (Klaus Biemann)
(A) Mass spectrometry applied to immunology:
Cells in the human body communicate their health status to the immune system by degrading cellular proteins and presenting fragments of each on the cell surface in association class I MHC proteins. Appropriately educated, cytotoxic T-lymphocytes (CTL) (CD8+ T-cells) bind to the class I MHC molecules on the cell surface, sample the protein fragments (peptides) being presented and kill those cells that express new peptides as a result of viral, bacterial and parasitic infection, tissue transplantation and cellular transformation (cancer). Since dysregulation of cell signaling pathways is one of the hallmarks of cancer, we hypothesized that class I MHC phosphopeptides that result from these pathways should be excellent candidates for use in the immunotherapy of cancer. Class I MHC phosphopeptides identified in preliminary work on leukemia, melanoma, and colorectal cancers elicit pre-existing, central-memory, T-cell-recall responses in multiple, healthy blood donors. Central memory recall responses to phosphopeptide antigens is absent in some leukemia patients and correlates with clinical outcome. The response is restored following allogenic stem cell transplantation. These results suggest strongly that class I MHC phosphopeptides are tumor targets of immune surveillance in humans and, therefore, are likely candidates for immunotherapy of cancer. Three of the discovered phosphopeptides will be the subject of a phase I clinical trial on melanoma later this spring. Proposed here is additional research to complete our studies on melanoma, leukemia, and colorectal cancer and to begin an effort to characterize class I MHC phosphopetides presented on esophageal, liver and ovarian cancer. Additional research will be conducted to identify the repertoire of altered class I or class II self-peptides that are induced by prescription drugs or environmental agents and lead to life threatening autoimmune disease.
B) Mass spectrometry applied characterization of protein post-translational modifications and antibody sequences.
Proposed here is research to develop a combination of mass spectrometry instrumentation and techniques plus chemical and biochemical methods that will facilitate identification and near complete amino acid sequence analysis of intact proteins or large protein fragments on a chromatographic time scale. This research will make it possible to characterize multiple post-translational modifications, particularly those that exist on the same protein molecule and together regulate its biological activity. This research is driven by five major innovations in my lab: development of (a) electron transfer dissociation (ETD) for fragmentation of intact proteins, (b) ETD and IIPT (ion-ion proton transfer) chemistry to obtain n- and c-terminal sequence information from intact proteins, (c) front end ETD (FETD) that facilitates a 10-50 fold increase in sensitivity for intact proteins, (d) micro-column enzyme reactors that generate 3-10 KDa proteins fragments and provide 96% sequence coverage for monoclonal antibodies and (e) methodology for enrichment of O-GlcNAcylated peptides by boronic acid chemistry in non-aqueous solvents. Going forward, we will implement new IIPT reagents to extend the usable mass range to m/z 4,000, employ a combination of IIPT and parallel ion parking strategies to concentrate multiple charge states observed in protein ESI into a single lower charge state for protein identification by CAD, develop parallel ion parking strategies to minimize second generation ETD reactions and thus allow us to read complete protein sequences from both the n- and c- termini of intact proteins, build a longer linear ion trap to increase the number of protein ions that can be stored for analysis, extend the micro-column enzyme reactor concept to other proteases and solvents and then apply all of this development to the analysis of proteins in outer membrane vesicles secreted by the antibiotic resistant, gram negative bacteria, Neisseria gonorrhoeae. The ultimate goal is to be able to identify bacterial, virulence-factor proteins on a chromatographic time scale. The above technology will also be employed to characterize (a) proteolytic cleavage events on histones that alter the epigenetic code, (b) O-GlcNAc sites on ribosomal proteins, mitochondrial proteins in the heart, and all known human kinases, and (c) protein binding partners for RSK1 (ribosomal protein S6 Kinase) that plays a major role in breast cancer.