List of Faculty
|
Donald
F. Hunt Analytical Biochemistry
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.
Representative Publications (from a list of 285)
Dynamic Regulation of HP1/Chromatin Interaction by Methylation and Phosphorylation of Histone H3, W. Fischle, B.S. Tseng, H. Dormann, B. M. Uberheide, B.A. Garcia, J. Shabanowitz, D.F. Hunt, H. Funabiki, and C.D. Allis, Nature, 2005, In Press.
Modifications of Human Histone H3 Isoforms During Mitosis, Garcis, B.A., Barber, C.M., Hake, S.B., Ptak, C., Turner, F.B., Busby, S.A., Shabanowitz, J., Moran, R.G., Allis, C.D., and Hunt, D.F., Biochemistry, 2005, 44, 13202 – 13213.
Nuclear Import of TFIIB is Mediated by Kap114P, a Karyopherin with Multiple Cargo-Binding domains, Hodges, J.L., Leslie, J.H., Mosammaparast, N., Guo, Y., Shabanowitz, J., Hunt, D.F., and Pemberton, L.F., Mol. Biol. Cell, 2005, 16, 3200 – 3210.
Analysis of Protein Phosphorylation by Mass Spectrometry, Garcia, B.A., Shabanowitz, J., and Hunt, D.F., Methods, 2005, 35, 256-264.
Protein Identification Using Sequential Ion/Ion Reactions and Tandem Mass Spectrometry, J.J. Coon, B. Ueberheide, J. E. P. Syka, D.D. Dryhurst, J. Ausio, J. Shabanowitz and D. F. Hunt, Proc. Natl., Acad. Sci., U.S.A., 2005, 102, 9463-9468.
Serine 31 Phosphorylation of Histone Variant H3.3 is Specific to Regions Bordering Centromeres in Metaphase Chromosomes, S.B. Hake, B.A. Garcia, M. Kauer, S.P. Baker, J. Shabanowitz, D.F. Hunt and C.D. Allis, Proc. Natl. Acad. Sci., U.S.A. 2005, 102, 6344 - 6349.
Mass Spectrometry Analysis of Arabidopsis Histone H3 Reveals Distinct Combinations of Post-Translational Modifications, L. Johnson, S. Mollah,T. L. Muratore, B.A. Garcia, J. Shabanowitz, D.F. Hunt, and Steve Jacobsen, Nucleic Acid Res., 2004, 32, 6511-6518.
Novel Linear Quadrupole Ion Trap/FT Mass Spectrometer: Performance Characterization and Use in the Comparative Analysis of Histone H3 Post-Translational Modifications, J.E.P Syka, J.A. Marto, D.L. Bai, S. Hornung, M.W. Senko, J.C. Schwartz, B. Ueberheide, B. Garcia, S. Busby, T. Muratore, J. Shabanowitz, and D.F. Hunt, J. Proteome Res., 2004, 3, 621-626.
Aurora B Phosphorylates Centromeric MCAK and Regulates Its Localization and Microtubule Depolymerization Activity, W. Lan, X. Zhang, S.L. Kline-Smith, S.E. Rosasco, G.A. Barrett-Wilt, J. Shabanowitz, D.F. Hunt, C.E. Walczak, and P.T. Stukenberg, Curr. Biol., 2004, 14, 273-286.
The Vertebrate Ndc80 Complex Contains Spc24 and Spc25 homologs, Which are Required to Establish and Maintain Kinetochore-Microtuble Attachment, M.L. McCleland, M.J. Kallio, G.A. Barrett-Wilt, C.A. Kestner, J. Shabanowitz, D.F. Hunt, G.J. Gorbsky, and P.T. Stukenberg, Curr. Biol., 2004, 14, 131-137.
Peptide and Protein Sequence Analysis by Electron Transfer Dissociation Mass Spectrometry, J.E. P. Syka, J.J. Coon, M.J. Schroeder, J. Shabanowitz, and D.F. Hunt, Proc. Natl. Acad. Sci., U.S.A., 2004, 101, 9528-9533.
Histone Methytransferases Direct Different Degrees of Methylation to Define Distinct Chromatin Domains, JC. Rice, S.D. Briggs, B. Uberheide, C. M. Barber, J. Shabanowitz, D.F. Hunt, Y. Shinkai T and C.D. Allis, Molecular Cell, 2003, 12, 1591-1598.
Identification of the Beta Cell Antigen Targeted by a Prevalent Population of Pathogenic CD8+ T Cells in Autoimmune Diabetes, S.M. Lieberman, A.M. Evans, B. Han, T. Takaki, Y. Vinnitskaya, J. Caldwell, D. Serreze, J. Shabanowitz, D.F. Hunt, S.G. Nathenson, P. Santamaria, and T.P. DiLorenzo, Proc. Natl. Acad. Sci., USA, 2003, 100, 8384-8388.
Gene Silencing:Trans-Histone Regulatory Pathway in Chrommatin, S.D. Briggs, T. Xiao, Z.-W. Sun, J.A. Caldwell, J. Shabanowitz, D.F. Hunt, C.D. Allis, B.D. Strahl, Nature, 2002, 418, 498.
The U3 Processome is a Large Nucleolar Ribonucleoprotein Required for 18S rRNA Biogenesis. F. Dragon, P.A. C. Post, J.E.G. Gallagher, B.M. Mitchell, K.A. Porwancher, K.A. Wehner, R.E. Settlage, J. Shabanowitz, Y. Osheim, A.L. Beyer, D.F. Hunt, and S.J. Baserga. Nature. 2002. 417: 967-970.
Phosphoproteome Analysis by Mass Spectrometry and its Application to Saccharomyces cerevisiae. S.B. Ficarro, M.L. McCleland, P.T. Stukenberg, D.J. Burke, M.M. Ross, J. Shabanowitz, D. F. Hunt, and F.M. White. Nature Biotechnology. 2002. 20: 301-305.
Identification and Modulation of a Naturally Processed T-Cell Epitope from the Diabetes-Associated Autoantigen 65 (hGAD65). G.T. Nepom, J. D. Lippolis, F. M.. White, S. Masewicz , J. A. Marto, A. Herman, C.J. Luckey, B. Falk, J. Shabanowitz , D. F. Hunt, and V. H. Engelhard, B. S. Nepom. Proc. Natl., Acad. Sci., USA. 2001. 98: 1763-1768.
Phosphorylated Peptides are Naturally Processed and Presented by MHC Class I Molecules In Vivo. A.L. Zarling, S.B. Ficarro, F.M. White, J.. Shabanowitz, D.F. Hunt, V.E. Engelhard. J. Exp. Med. 2001. 192: 1755-1762.
Mitotic Phosphorylation of Histone H3 is Governed by lpl1/Aurora Kinase and Glc7/PP1 Phosphatase in Budding Yeast and Nematodes. J-Yuan Hsu, Z-W. Sun, X.Li, M. Reuben, K. Tatchell, D.K. Bishop, J.M. Grushcow, C.J. Brame, J.A. Caldwell, D.F. Hunt, R. Lin, M.M. Smith and C.D. Allis. Cell. 2000. 102: 279-291.
Sub-Femtomole MS and MS/MS Peptide Sequence Analysis Using LC-Nano-ESI Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. S.E. Martin, J. Shabanowitz, D.F. Hunt, and J.A. Marto. Anal. Chem. 2000. 72: 4266-4274.
A Myosin I Isoform in the Nucleus. L. Pestic-Dragovich, L.Stojiljkovic, A.A. Philimonenko, G. Nowak, Y. Ke, R. E. Settlage, J. Shabanowitz, D.F. Hunt, P. Hozak, and P. de Lanerolle. Science. 2000. 290: 337-341.
The A1-HY Minor Histocompatibility Antigen Originates From DFFRY and Contains a Cysteinylated Cysteine Residue as Identified by a Novel Mass Spectrometric Technique, R.A. Pierce, E.D. Field, J.M.M. den Haan, J.A. Caldwell, F. M. White, J.A. Marto, J. Shabanowitz, D. F. Hunt, Els Goulmy and V. H. Engelhard, J. Immunol., 1999, 163, 6360-6364.
Biochemical Identification of a Mutated Human Melanoma Antigen Recognized by CD4+ T Cells, R. Pieper, R. Christian, M.I. Gonzales, M.I. Nishimura, G. Gupta, S.A. Rosenberg, D.F. Hunt, and S.L. Topalian, J. Exp. Med., 1999, 189, 757-765.
The Class I Antigen-Processing Pathway for the Membrane Protein Tyrosinase Involves Translation in the Endoplasmic Reticulum and Processing in the Cytosol, C.A. Mosse, L. Meadows, C.J. Luckey, D.J. Kittlesen, E.L. Huczko, C.L. Slingluff, J. Shabanowitz, D.F. Hunt, and V.H. Engelhard, J. Exp. Med., 1998, 187, 37-48.
The Minor Histocompatibility Antigen HA-1: A Diallelic Gene with a Single Amino Acid Polymorphism. J.M.M. den Haan, L.M. Meadows, W. Wang, J.Pool, E.Blokland, T.L. Bishop, C. Reinhardus, J. Shabanowitz, R. Offringa, D.F. Hunt, V. E. Engelhard, and E. Goulmy. Science. 1998. 279: 1054-1057.
The HLA-A*0201 H-Y Antigen Contains a Posttranslationally Modified Cysteine That Significantly Affects T Cell Recognition, L. Meadows, W. Wang, J.M.M. den Haan, E. Blokland, C. Reinhardus, J.W. Driijfhout, J. Shabanowitz, R. Pierce, A.I. Agulnik, C.E. Bishop, D.F. Hunt, E. Goulmy, and V.H. Engelhard, Immunity, 1997, 6, 273-281.
Specific Recognition of Thymic Self-Peptides Induces the Positive Selection of Cytotoxic T Lymphocytes, Q. Hu, C.R.B. Walker, C. Girao, J.T. Opferman, J. Sun, J. Shabanowitz, D.F. Hunt, and P.G. Ashton-Rickardt, Immunity, 1997, 7, 221-231.
The Proteolytic Fragments Generated by Vertebrate Proteasomes: Structural Relationships to MHC Class I Binding Peptides, G. Niedermann, G. King, S. Butz, U. Birsner, R. Grimm. J. Shabanowitz, D.F. Hunt, and K. Eichmann, Proc. Natl. Acad. Sci., USA 1996, 93, 8572-8577.
The Immunodominant Major Histocompatibility Complex class I-Restricted Antigen of a Murine Colon tumor Derives from an Endogenous Retroviral Gene Product, A. Y. C. Wang, P.H. Gulden, A.S. Woods, M.C. Thomas, C.D. Tong, W. Wang, V.H. Engelhard, G. Pasternack, R. Cotter, D.F. Hunt, D.M. Pardoll, and E.M. Jaffe, Proc. Natl, Acad. Sci., USA, 1996, 93, 9730-9735.
Identification of a Graft-Versus-Host Disease Associated Human Minor Histocompatibility Antigen. J.M.M. den Haan, N.E. Sherman, E. Blokland, E. Huczko, F. Konig, J. W. Drijhout, J. Skipper, J. Shabanowitz, D.F. Hunt, V.H. Engelhard and E. Goulmy. Science. 1995. 268: 1476-1480.
Human H-Y: A Male Specific Histocompatibility Antigen Derived from the SMCY Protein. W. Wang, L.R.Meadows, J.M.M. dan Haan, N.E. Sherman, Y. Chen, E. Blokland, J. Shabanowitz, A. I.Agulnik, R.C. Hendrickson, C.E. Bishop, D.F. Hunt, E. Goulmy,and V.H. Engelhard. Science. 1995. 269: 1588-1590.
Identification of a Peptide Recognized by Five Melanoma Specific Human Cytotoxic T-Cell Lines. A.L. Cox, J. Skipper, Y. Chen, R.A. Henderson, T.L. Darrow, J. Shabanowitz, V.H. Engelhard, D.F. Hunt, and C.L. Slingluff, Jr. Science. 1994. 264: 716-719.
Internal Lysine Palmitolylation in Adenylate Cyclase Toxin from Bordetella Pertussis. M. Hackett, L. Guo, J. Shabanowitz, D.F. Hunt, E.L. Hewlett, Science. 1994. 266: 433-435.
Direct Identification of an Endogeneous Peptide Recognized by Multiple HLA-A2.1 Specific Cytotoxic T Cells, R.A. Henderson, A. L. Cox, K. Sakaguichi, E. Appella, J. Shabanowitz, D.F. Hunt and V.H. Engelhard, Proc. Natl. Acad. Sci., U.S.A., 1993, 90, 10275-10279.
Characterization of Peptides Associated with the Class I MHC Molecule, HLA-A2.1. D.F. Hunt, R. A. Henderson, J. Shabanowitz, K. Sakaguchi, H. Michel, N. Sevilir, A.L. Cox, E. Appella, and V. H. Engelhard. Science. 1992. 255: 1261-1263.
HLA-A2.1 Associated Peptides From a Mutant Cell Line: A Second Pathway of Antigen Presentation. R.A. Henderson, H. Michel, K. Sakaguchi, J. Shabanowitz, E. Appella, D.F. Hunt and V.H. Engelhard. Science. 1992. 255: 1264-1266.
Peptides Presented to the Immune System by the Murine Class II MHC Molecule, IAd. D.F. Hunt, H. Michel, T.A. Dickinson, J. Shabanowitz, A.L. Cox, K. Sakaguchi, E. Appella, H. Grey and A. Sette. Science. 1992. 256: 1817-1820.
Invariant Chain Peptides in Most HLA-DR Molecules of an Antigen Processing Mutant. A. Sette, S. Ceman, R.T. Kubo, K. Sakaguchi, E. Appella, D.F. Hunt, T. A. Davis, H. Michel, J. Shabanowitz, R. Rudersdorf, H. M. Grey, and R. DeMars. Science. 1992. 285: 1801-1804.