Donald F. Hunt, PhD

Donald F. Hunt, PhD

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Application of mass spectrometry to identification of peptide antigens and proteins in complex mixtures

Protein phosphorylation is known to be dysregulated in cancer cells and phosphopeptides have been found on the cell surface in association with a variety of human class I MHC molecules including HLA A2.1, B7, B8 and B27 (A.L. Zarling, S.B. Ficarro, F.M. White, J. Shabanowitz, D.F. Hunt, V.E. Engelhard, J. Exp. Med., 2001, 192, 1755-1762).  These observations led them to test the hypothesis that some phosphopeptides might be unique to cancer cells and able to stimulate an immune response that would irradicate the tumor.  In short, phosphopeptides uniquely found on tumors in association with MHC molecules might become diagnostic of the particular cancer, be used to stimulate a therapeutic response and even to vaccinate a population against a particular cancer.  Phosphopeptides found to be shared uniquely by multiple types of cancer cells would have even higher value. To date, they have sequenced 16 phosphopeptides presented by HLA-A2.1 on the EBV-transformed B-lymphoblastoid (control) JY cell line.  All sixteen peptides were detected in multiple preparations and found to be present at levels above 10 copies/cell.  In the case of the cancer analysis, a total of 17 phosphopeptides have been detected thus far on the surface of melanoma and ovarian cancer cells. Six are also found on the control JY cell line. Among the other 11 phosphopeptides, 3 are uniquely expressed on the two melanoma cell lines, 5 are unique to a single melanoma cell line and 3 are common to all cancer lines but not in the control JY.  Source proteins for these differentially expressed phosphopeptides have all been implicated in either cancer or cell cycle checkpoint dysregulation.  Western blots against the source proteins confirm their absence in the JY control cell line.  Cytotoxic T-cells against two of the above peptides have been generated and shown to lyse both peptide pulsed cells and the original cancer cell line.  Efforts are underway to generate cytotoxic T-cells against all differentially expressed phosphopeptides.  Once generated, these T-cells can be used as reagents to check for the presence of the individual phosphopeptides on the surface of additional cell types, both normal and derived from tumor.   This work is being extended to colon, ovarian and breast cancer and to cells presenting MHC molecules such as A1, A3, B7, B8 and B44.  Electron transfer dissociation (ETD) (see two articles above) is being used for these studies.  In addition, they have developed new methodology that used isotopic labeling in combination with immobilized metal affinity chromatography (IMAC) to conduct a rapid survey of tumor samples to confirm that the same phosphopeptide epitopes are presented on multiple tumors of the same type and thus shared by all persons who have the particular cancer and express the appropriated MHC molecule.