Donald F. Hunt


  • BS, University of Massachusetts, Amherst, MA
  • PhD, University of Massachusetts, Amherst, MA
  • Postdoc, MIT, Cambridge, MA (Klaus Biemann)

Primary Appointment

  • Professor, Chemistry


Research Interest(s)

Analytical Biochemistry

Research Description

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.

Selected Publications

  • Papazyan R, Voronina E, Chapman J, Luperchio T, Gilbert T, Meier E, Mackintosh S, Shabanowitz J, Tackett A, Reddy K, Coyne R, Hunt D, Liu Y, Taverna S. Methylation of histone H3K23 blocks DNA damage in pericentric heterochromatin during meiosis. eLife. 2014;3 e02996. PMID: 25161194 | PMCID: PMC4141274
  • Bailey A, Panchenko T, Sathyan K, Petkowski J, Pai P, Bai D, Russell D, Macara I, Shabanowitz J, Hunt D, Black B, Foltz D. Posttranslational modification of CENP-A influences the conformation of centromeric chromatin. Proceedings of the National Academy of Sciences of the United States of America. 2013;110(29): 11827-32. PMID: 23818633 | PMCID: PMC3718089
  • Petkowski J, Schaner Tooley C, Anderson L, Shumilin I, Balsbaugh J, Shabanowitz J, Hunt D, Minor W, Macara I. Substrate Specificity of Mammalian N-Terminal ╬▒-Amino Methyltransferase NRMT. Biochemistry. 2012;51(30): 5942-50. PMID: 22769851 | PMCID: PMC3447998
  • Wu D, Chapman J, Wang L, Harris T, Shabanowitz J, Hunt D, Fu Z. Intestinal cell kinase (ICK) promotes activation of mTOR complex 1 (mTORC1) through phosphorylation of Raptor Thr-908. The Journal of biological chemistry. 2012;287(15): 12510-9. PMID: 22356909 | PMCID: PMC3321000
  • Compton P, Strukl J, Bai D, Shabanowitz J, Hunt D. Optimization of electron transfer dissociation via informed selection of reagents and operating parameters. Analytical chemistry. 2011;84(3): 1781-5. PMID: 22182179 | PMCID: PMC3277643
  • Baker S, Phillips J, Anderson S, Qiu Q, Shabanowitz J, Smith M, Yates J, Hunt D, Grant P. Histone H3 Thr 45 phosphorylation is a replication-associated post-translational modification in S. cerevisiae. Nature cell biology. 2010;12(3): 294-8. PMID: 20139971 | PMCID: PMC2856316
  • Gordon V, Bhadel S, Wunderlich W, Zhang J, Ficarro S, Mollah S, Shabanowitz J, Hunt D, Xenarios I, Hahn W, Conaway M, Carey M, Gioeli D. CDK9 regulates AR promoter selectivity and cell growth through serine 81 phosphorylation. Molecular endocrinology (Baltimore, Md.). 2010;24(12): 2267-80. PMID: 20980437 | PMCID: PMC2999477
  • Hall E, Balsbaugh J, Rose K, Shabanowitz J, Hunt D, Brautigan D. Comprehensive analysis of phosphorylation sites in Tensin1 reveals regulation by p38MAPK. Molecular & cellular proteomics : MCP. 2010;9(12): 2853-63. PMID: 20798394 | PMCID: PMC3101865
  • Hao Y, Du Q, Chen X, Zheng Z, Balsbaugh J, Maitra S, Shabanowitz J, Hunt D, Macara I. Par3 controls epithelial spindle orientation by aPKC-mediated phosphorylation of apical Pins. Current biology : CB. 2010;20(20): 1809-18. PMID: 20933426 | PMCID: PMC2963683
  • Tooley C, Petkowski J, Muratore-Schroeder T, Balsbaugh J, Shabanowitz J, Sabat M, Minor W, Hunt D, Macara I. NRMT is an alpha-N-methyltransferase that methylates RCC1 and retinoblastoma protein. Nature. 2010;466(7310): 1125-8. PMID: 20668449 | PMCID: PMC2939154
  • Wang Z, Udeshi N, Slawson C, Compton P, Sakabe K, Cheung W, Shabanowitz J, Hunt D, Hart G. Extensive crosstalk between O-GlcNAcylation and phosphorylation regulates cytokinesis. Science signaling. 2010;3(104): ra2. PMID: 20068230 | PMCID: PMC2866299
  • Basu A, Rose K, Zhang J, Beavis R, Ueberheide B, Garcia B, Chait B, Zhao Y, Hunt D, Segal E, Allis C, Hake S. Proteome-wide prediction of acetylation substrates. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(33): 13785-90. PMID: 19666589 | PMCID: PMC2728972
  • Depontieu F, Qian J, Zarling A, McMiller T, Salay T, Norris A, English A, Shabanowitz J, Engelhard V, Hunt D, Topalian S. Identification of tumor-associated, MHC class II-restricted phosphopeptides as targets for immunotherapy. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(29): 12073-8. PMID: 19581576 | PMCID: PMC2715484
  • Ishibashi T, Dryhurst D, Rose K, Shabanowitz J, Hunt D, Ausi├│ J. Acetylation of vertebrate H2A.Z and its effect on the structure of the nucleosome. Biochemistry. 2009;48(22): 5007-17. PMID: 19385636 | PMCID: PMC2850812
  • Shechter D, Chitta R, Xiao A, Shabanowitz J, Hunt D, Allis C. A distinct H2A.X isoform is enriched in Xenopus laevis eggs and early embryos and is phosphorylated in the absence of a checkpoint. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(3): 749-54. PMID: 19131518 | PMCID: PMC2630098
  • Wang Z, Udeshi N, O'Malley M, Shabanowitz J, Hunt D, Hart G. Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass spectrometry. Molecular & cellular proteomics : MCP. 2009;9(1): 153-60. PMID: 19692427 | PMCID: PMC2808261
  • Bernstein E, Muratore-Schroeder T, Diaz R, Chow J, Changolkar L, Shabanowitz J, Heard E, Pehrson J, Hunt D, Allis C. A phosphorylated subpopulation of the histone variant macroH2A1 is excluded from the inactive X chromosome and enriched during mitosis. Proceedings of the National Academy of Sciences of the United States of America. 2008;105(5): 1533-8. PMID: 18227505 | PMCID: PMC2234179
  • Duncan E, Muratore-Schroeder T, Cook R, Garcia B, Shabanowitz J, Hunt D, Allis C. Cathepsin L proteolytically processes histone H3 during mouse embryonic stem cell differentiation. Cell. 2008;135(2): 284-94. PMID: 18957203 | PMCID: PMC2579750
  • Eisinger-Mathason T, Andrade J, Groehler A, Clark D, Muratore-Schroeder T, Pasic L, Smith J, Shabanowitz J, Hunt D, Macara I, Lannigan D. Codependent functions of RSK2 and the apoptosis-promoting factor TIA-1 in stress granule assembly and cell survival. Molecular cell. 2008;31(5): 722-36. PMID: 18775331 | PMCID: PMC2654589
  • Housley M, Rodgers J, Udeshi N, Kelly T, Shabanowitz J, Hunt D, Puigserver P, Hart G. O-GlcNAc regulates FoxO activation in response to glucose. The Journal of biological chemistry. 2008;283(24): 16283-92. PMID: 18420577 | PMCID: PMC2423255
  • Mohammed F, Cobbold M, Zarling A, Salim M, Barrett-Wilt G, Shabanowitz J, Hunt D, Engelhard V, Willcox B. Phosphorylation-dependent interaction between antigenic peptides and MHC class I: a molecular basis for the presentation of transformed self. Nature immunology. 2008;9(11): 1236-43. PMID: 18836451 | PMCID: PMC2596764
  • Prickett T, Ninomiya-Tsuji J, Broglie P, Muratore-Schroeder T, Shabanowitz J, Hunt D, Brautigan D. TAB4 stimulates TAK1-TAB1 phosphorylation and binds polyubiquitin to direct signaling to NF-kappaB. The Journal of biological chemistry. 2008;283(28): 19245-54. PMID: 18456659 | PMCID: PMC2443674
  • Rowlett R, Chrestensen C, Schroeder M, Harp M, Pelo J, Shabanowitz J, DeRose R, Hunt D, Sturgill T, Worthington M. Inhibition of tristetraprolin deadenylation by poly(A) binding protein. American journal of physiology. Gastrointestinal and liver physiology. 2008;295(3): G421-30. PMID: 18467502 | PMCID: PMC2536786
  • Calvert M, Keck K, Ptak C, Shabanowitz J, Hunt D, Pemberton L. Phosphorylation by casein kinase 2 regulates Nap1 localization and function. Molecular and cellular biology. 2007;28(4): 1313-25. PMID: 18086883 | PMCID: PMC2258750
  • Chen T, Muratore T, Schaner-Tooley C, Shabanowitz J, Hunt D, Macara I. N-terminal alpha-methylation of RCC1 is necessary for stable chromatin association and normal mitosis. Nature cell biology. 2007;9(5): 596-603. PMID: 17435751
  • Chi A, Bai D, Geer L, Shabanowitz J, Hunt D. Analysis of intact proteins on a chromatographic time scale by electron transfer dissociation tandem mass spectrometry. International journal of mass spectrometry. 2007;259(1): 197-203. PMID: 17364019 | PMCID: PMC1826913
  • Frum R, Busby S, Ramamoorthy M, Deb S, Shabanowitz J, Hunt D, Deb S. HDM2-binding partners: interaction with translation elongation factor EF1alpha. Journal of proteome research. 2007;6(4): 1410-7. PMID: 17373842
  • Goldfinger L, Ptak C, Jeffery E, Shabanowitz J, Han J, Haling J, Sherman N, Fox J, Hunt D, Ginsberg M. An experimentally derived database of candidate Ras-interacting proteins. Journal of proteome research. 2007;6(5): 1806-11. PMID: 17439166
  • Hunt D, Henderson R, Shabanowitz J, Sakaguchi K, Michel H, Sevilir N, Cox A, Appella E, Engelhard V. Pillars article: Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry. Science 1992. 255: 1261-1263. Journal of immunology (Baltimore, Md. : 1950). 2007;179(5): 2669-71. PMID: 17709476
  • Luo W, Peterson A, Garcia B, Coombs G, Kofahl B, Heinrich R, Shabanowitz J, Hunt D, Yost H, Virshup D. Protein phosphatase 1 regulates assembly and function of the beta-catenin degradation complex. The EMBO journal. 2007;26(6): 1511-21. PMID: 17318175 | PMCID: PMC1829374
  • Mayhew M, Jeffery E, Sherman N, Nelson K, Polefrone J, Pratt S, Shabanowitz J, Parsons J, Fox J, Hunt D, Horwitz A. Identification of phosphorylation sites in betaPIX and PAK1. Journal of cell science. 2007;120 3911-8. PMID: 17989089
  • Taverna S, Ueberheide B, Liu Y, Tackett A, Diaz R, Shabanowitz J, Chait B, Hunt D, Allis C. Long-distance combinatorial linkage between methylation and acetylation on histone H3 N termini. Proceedings of the National Academy of Sciences of the United States of America. 2007;104(7): 2086-91. PMID: 17284592 | PMCID: PMC1892956
  • Udeshi N, Shabanowitz J, Hunt D, Rose K. Analysis of proteins and peptides on a chromatographic timescale by electron-transfer dissociation MS. The FEBS journal. 2007;274(24): 6269-76. PMID: 18021239
  • Altrich-VanLith M, Ostankovitch M, Polefrone J, Mosse C, Shabanowitz J, Hunt D, Engelhard V. Processing of a class I-restricted epitope from tyrosinase requires peptide N-glycanase and the cooperative action of endoplasmic reticulum aminopeptidase 1 and cytosolic proteases. Journal of immunology (Baltimore, Md. : 1950). 2006;177(8): 5440-50. PMID: 17015730
  • Auphan-Anezin N, Mazza C, Guimezanes A, Barrett-Wilt G, Montero-Julian F, Roussel A, Hunt D, Malissen B, Schmitt-Verhulst A. Distinct orientation of the alloreactive monoclonal CD8 T cell activation program by three different peptide/MHC complexes. European journal of immunology. 2006;36(7): 1856-66. PMID: 16761314
  • Brickner A, Evans A, Mito J, Xuereb S, Feng X, Nishida T, Fairfull L, Ferrell R, Foon K, Hunt D, Shabanowitz J, Engelhard V, Riddell S, Warren E. The PANE1 gene encodes a novel human minor histocompatibility antigen that is selectively expressed in B-lymphoid cells and B-CLL. Blood. 2006;107(9): 3779-86. PMID: 16391015 | PMCID: PMC1895781
  • Chi A, Valencia J, Hu Z, Watabe H, Yamaguchi H, Mangini N, Huang H, Canfield V, Cheng K, Yang F, Abe R, Yamagishi S, Shabanowitz J, Hearing V, Wu C, Appella E, Hunt D. Proteomic and bioinformatic characterization of the biogenesis and function of melanosomes. Journal of proteome research. 2006;5(11): 3135-44. PMID: 17081065
  • Garcia B, Shabanowitz J, Hunt D. Characterization of histones and their post-translational modifications by mass spectrometry. Current opinion in chemical biology. 2006;11(1): 66-73. PMID: 17157550
  • Goldfinger L, Ptak C, Jeffery E, Shabanowitz J, Hunt D, Ginsberg M. RLIP76 (RalBP1) is an R-Ras effector that mediates adhesion-dependent Rac activation and cell migration. The Journal of cell biology. 2006;174(6): 877-88. PMID: 16966426 | PMCID: PMC2064341
  • Harris T, Chi A, Shabanowitz J, Hunt D, Rhoads R, Lawrence J. mTOR-dependent stimulation of the association of eIF4G and eIF3 by insulin. The EMBO journal. 2006;25(8): 1659-68. PMID: 16541103 | PMCID: PMC1440840
  • Harris T, Huffman T, Chi A, Shabanowitz J, Hunt D, Kumar A, Lawrence J. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1. The Journal of biological chemistry. 2006;282(1): 277-86. PMID: 17105729
  • Herlevsen M, Oxford G, Ptak C, Shabanowitz J, Hunt D, Conaway M, Theodorescu D. A novel model to identify interaction partners of the PTEN tumor suppressor gene in human bladder cancer. Biochemical and biophysical research communications. 2006;352(2): 549-55. PMID: 17126809 | PMCID: PMC1933505
  • Li Y, Kao G, Garcia B, Shabanowitz J, Hunt D, Qin J, Phelan C, Lazar M. A novel histone deacetylase pathway regulates mitosis by modulating Aurora B kinase activity. Genes & development. 2006;20(18): 2566-79. PMID: 16980585 | PMCID: PMC1578679
  • Lin C, Platt M, Ficarro S, Hoofnagle M, Shabanowitz J, Comai L, Hunt D, Owens G. Mass spectrometric identification of phosphorylation sites of rRNA transcription factor upstream binding factor. American journal of physiology. Cell physiology. 2006;292(5): C1617-24. PMID: 17182730
  • Martin K, Jeffery E, Grigera P, Shabanowitz J, Hunt D, Parsons J. Cortactin phosphorylation sites mapped by mass spectrometry. Journal of cell science. 2006;119 2851-3. PMID: 16825425
  • Morris S, Rao B, Garcia B, Hake S, Diaz R, Shabanowitz J, Hunt D, Allis C, Lieb J, Strahl B. Identification of histone H3 lysine 36 acetylation as a highly conserved histone modification. The Journal of biological chemistry. 2006;282(10): 7632-40. PMID: 17189264 | PMCID: PMC2811852
  • Mosoian A, Teixeira A, High A, Christian R, Hunt D, Shabanowitz J, Liu X, Klotman M. Novel function of prothymosin alpha as a potent inhibitor of human immunodeficiency virus type 1 gene expression in primary macrophages. Journal of virology. 2006;80(18): 9200-6. PMID: 16940531 | PMCID: PMC1563913
  • Webb D, Mayhew M, Kovalenko M, Schroeder M, Jeffery E, Whitmore L, Shabanowitz J, Hunt D, Horwitz A. Identification of phosphorylation sites in GIT1. Journal of cell science. 2006;119 2847-50. PMID: 16825424
  • Zarling A, Polefrone J, Evans A, Mikesh L, Shabanowitz J, Lewis S, Engelhard V, Hunt D. Identification of class I MHC-associated phosphopeptides as targets for cancer immunotherapy. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(40): 14889-94. PMID: 17001009 | PMCID: PMC1595446