REGULAR ARTICLE T cell epitope definition by differential mass spectrometry: Identification of a novel, immunogenic HLA-B8 ligand directly from renal cancer tissue Thomas Flad 1 , Ludmila Mueller 1 , Hassan Dihazi 2 , Veneta Grigorova 1 , Ralf Bogumil 3 , Alexander Beck 4 , Cornelia Thedieck 1 , Gerhard A. Mueller 2 , Hubert Kalbacher 5 and Claudia A. Mueller 1 1 Section for Transplantation Immunology and Immunohematology, University of Tuebingen, Tuebingen, Germany 2 Department of Rheumatology and Nephrology, University of Goettingen, Goettingen, Germany 3 Ciphergen Biosystems GmbH, Goettingen, Germany 4 Clinical-chemical Central Laboratory, Department of Internal Medicine IV, University of Tuebingen, Tuebingen, Germany 5 Medical and Natural Sciences ResearchCenter, University of Tuebingen, Tuebingen, Germany In this study, we describe a differential mass spectrometric technique for the immuno-proteomic analysis of the major histocompatibility complex (MHC) peptides of a renal cell carcinoma (RCC) biopsy compared with the healthy kidney tissue of the same patient after nephrectomy. Using a stable isotope labeling approach, we could directly compare and relatively quantify 43 MHC-peptide pairs, most of which were present in similar proportions on both normal kidney and tumor. Sig- nificantly, two dominant peptides of monoisotopic masses ([M1H] 1 ) 973.43 u and 967.59 u, respectively, were found exclusively in the tumor sample. One of these was identified as originating from heme oxygenase-1 (HO-1), a protein involved in induction of apoptosis resistance, immuno- suppression and neoangiogenesis and reported to be up-regulated in various cancer types. Moreover, the corresponding synthetic HO-1-derived peptide was shown to be immunogenic in vitro by gen- eration of CD8 1 T cell lines with peptide-specific cytolytic activity. Thus, this peptide is an example of a differentially identified T cell epitope that could be considered as a target for immunotherapy. Received: February 21, 2005 Revised: April 4, 2005 Accepted: May 10, 2005 Keywords: Major histocompatibility complex / Mass spectrometry / Renal cell carcinoma / Stable isotope labeling / Tumor antigen Proteomics 2006, 5, 0000–0000 1 1 Introduction The definition of novel tumor-specific MHC-peptides as tar- gets for cytotoxic T lymphocytes (CTL) will provide greater scope for targeted immunotherapies against cancer. So far, the identification of such peptides has been hindered by the presence of only a small number of tumor-specific peptides amongst several thousands of different MHC-peptides on each tumor cell. The identification of human tumor T cell antigens has to a great extent relied on the use of specific T lymphocytes recognizing the tumor, either based on a gene expression approach [1] or on mass spectrometric sequenc- ing of isolated MHC-peptides [2]. At the time of writing, the public SYFPEITHI database (www.syfpeithi.de) lists about 200 identified human MHC-class I peptides, classified as “cancer-related”. Most of these peptides were identified by experiments based on their recognition by tumor-specific Correspondence: Dr. Thomas Flad, ZMF, Waldhoernlestr. 22, 72072 Tuebingen, Germany E-mail: thomas.flad@gmx.de Fax: 149-7071-295567 Abbreviation: RCC, renal cell carcinoma  2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de DOI 10.1002/pmic.200500099