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

Two areas:

(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.

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
  • Abreu C, Kumar R, Hamilton D, Dawdy A, Creavin K, Eivers S, Finn K, Balsbaugh J, O'Connor R, Kiely P, Shabanowitz J, Hunt D, Grenon M, Lowndes N. Site-specific phosphorylation of the DNA damage response mediator rad9 by cyclin-dependent kinases regulates activation of checkpoint kinase 1. PLoS genetics. 2013;9(4): e1003310. PMID: 23593009 | PMCID: PMC3616908
  • 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
  • Earley L, Anderson L, Bai D, Mullen C, Syka J, English A, Dunyach J, Stafford G, Shabanowitz J, Hunt D, Compton P. Front-end electron transfer dissociation: a new ionization source. Analytical chemistry. 2013;85(17): 8385-90. PMID: 23909443 | PMCID: PMC3822909
  • Mothé B, Southwood S, Sidney J, English A, Wriston A, Hoof I, Shabanowitz J, Hunt D, Sette A. 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. Immunogenetics. 2013;65(5): 371-86. PMID: 23417323 | PMCID: PMC3633659
  • Pérez J, Udeshi N, Shabanowitz J, Ciordia S, Juárez S, Scott C, Olszewski N, Hunt D, García J. O-GlcNAc modification of the coat protein of the potyvirus Plum pox virus enhances viral infection. Virology. 2013;442(2): 122-31. PMID: 23639873
  • 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 α-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
  • 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, Ausió 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
  • 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
  • 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