The goal of our research is to develop new methods and instrumentation for the structural characterization of proteins and their post translational modifications at the low femtomole/attomole level and to apply these new methods to important structural problems in cell biology and immunology. Towards this end, we have pioneered the use of nanoflow HPLC in conjunction with microelectrospray ionization on ion trap and Fourier transform mass spectrometers. Briefly stated, the approach involves the use of proteolytic enzymes to convert the protein or group of proteins into a complex mixture of peptides, which are then fractionated by nanoflow-HPLC and eluted directly into the mass spectrometer. Mixtures containing thousands of different peptides can be analyzed in this manner. Protonated peptides of a particular mass are selected under computer control of instrument, fragmented on collision with helium atoms and the resulting fragments are then separated and mass analyzed. Dissociation of the peptide ions occurs more or less randomly at each of the amide bonds in the molecules to produce a collection of fragments. The mass difference between two fragments differing by a single amino acid defines the mass and thus the identity of the extra residue in the longer fragment. Peptide sequence analysis is performed routinely at the femtomole and low attomole levels on the ion trap and Fourier transform instruments, respectively. Mass spectra acquired in the above manner can also be used to search databases and to identify known proteins. Currently, this approach is the most sensitive method in the world for protein characterization.
Our research focuses on two major applications of the above technology. The first involves identifying peptides that trigger the immune system to kill diseased cells. Cytotoxic T lymphocytes (CTL) or killer cells are an arm of the immune system concerned with recognition of cells that express new antigens, proteins, as a result of viral infection or cellular transformation (cancer). Cells convey their health status to the immune system by generating fragments from each of the approximately 10,000 proteins being synthesized, loading them onto a protein carrier (MHC molecule), and transporting them to the cell surface for screening by the killer cells. CTL lyse those cells that display new fragments, antigens that are associated with a particular disease state. Identification of these antigens is the first step in the preparation of vaccines that promote immunity against the above diseases. Peptides that cause the immune system to kill melanoma and lung cancer cells, to reject bone marrow transplants to leukemia patients, and to lyse tuberculosis infected cells have been identified in the laboratory recently. Efforts are in progress to characterize the peptide antigens that (a) cause rejection of tissue transplants, (b) trigger organ or tissue destruction in such autoimmune disorders as diabetes, arthritis, and multiple sclerosis, and (c) initiate an immunological response to breast, ovarian, colon-rectal, lung, and prostate cancers.
The second application involves research in the field of proteomics. DNA sequence information on the human genone and that of selected organisms is now becoming available at an ever increasing rate and will provide the starting point for development of novel therapeutic interventions against many of the world’s diseases. The next challenge is at the level of proteomics, understanding the functions of proteins encoded by a particular genome. Presently under development are mass spectrometry methods that will facilitate differential display and quantitation of most, if not all, proteins expressed by healthy vs diseased cells or cells grown in the presence or absence of drugs or other agonists. Mass spectrometry is also being used to analyze all proteins secreted by a particular cell type, to identify components of functionally active protein complexes, to probe protein-protein and protein-DNA interactions, and to locate post translational modifications and covalently attached ligands. Recently, we have developed methods that facilitate analysis of all phosphoproteins expressed in a particular cell population.
Front-End Electron Transfer Dissociation: A New Ionization Source. Earley L, Anderson LC, Bai DL, Mullen C, Syka JE, English AM, Dunyach JJ, Stafford GC Jr, Shabanowitz J, Hunt DF, Compton PD. Anal Chem. 2013 Aug 19. [Epub ahead of print]
Posttranslational modification of CENP-A influences the conformation of centromeric chromatin. Bailey AO, Panchenko T, Sathyan KM, Petkowski JJ, Pai PJ, Bai DL, Russell DH, Macara IG, Shabanowitz J, Hunt DF, Black BE, Foltz DR. Proc Natl Acad Sci U S A. 110:11827-32 (2013).
O-GlcNAc modification of the coat protein of the potyvirus Plum pox virus enhances viral infection. Pérez Jde J, Udeshi ND, Shabanowitz J, Ciordia S, Juárez S, Scott CL, Olszewski NE, Hunt DF, García JA. Virology. 2442:122-31 (2013).
Site-specific phosphorylation of the DNA damage response mediator rad9 by cyclin-dependent kinases regulates activation of checkpoint kinase 1. Abreu CM, Kumar R, Hamilton D, Dawdy AW, Creavin K, Eivers S, Finn K, Balsbaugh JL, O’Connor R, Kiely PA, Shabanowitz J, Hunt DF, Grenon M, Lowndes NF. PLoS Genet. 9:e1003310 (2013).
Peptide-binding motifs associated with MHC molecules common in Chinese rhesus macaques are analogous to those of human HLA supertypes and include HLA-B27-like alleles. Mothé BR, Southwood S, Sidney J, English AM, Wriston A, Hoof I, Shabanowitz J, Hunt DF, Sette A. Immunogenetics. 65:371-86 (2013).
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