Hindawi Publishing Corporation International Journal of Proteomics Volume 2011, Article ID 628787, 13 pages doi:10.1155/2011/628787 Research Article The Application of a Three-Step Proteome Analysis for Identification of New Biomarkers of Pancreatic Cancer Mayinuer Abulaizi, 1, 2 Takeshi Tomonaga, 1, 3 Mamoru Satoh, 1, 2 Kazuyuki Sogawa, 1, 2 Kazuyuki Matsushita, 1, 2 Yoshio Kodera, 2, 4 Jurat Obul, 1 Shigetsugu Takano, 5 Hideyuki Yoshitomi, 5 Masaru Miyazaki, 5 and Fumio Nomura 1, 2 1 Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan 2 Clinical Proteomics Research Center, Chiba University Hospital, Chiba 260-8677, Japan 3 Laboratory of Proteome Research, National Institute of Biomedical Innovation, Osaka 567-0085, Japan 4 Laboratory of Biomolecular Dynamics, Department of Physics, Kitasato University School of Science, Sagamihara 252-0373, Japan 5 Department of General Surgery, Graduate School of Medicine, Chiba University, 260-8670 Chiba, Japan Correspondence should be addressed to Mayinuer Abulaizi, ablizmaynur@gmail.com Received 31 May 2011; Accepted 2 August 2011 Academic Editor: Tadashi Kondo Copyright © 2011 Mayinuer Abulaizi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. We searched for novel tumor markers of pancreatic cancer by three-step serum proteome analysis. Twelve serum abundant proteins were depleted using immunoaffinity columns followed by fractionation by reverse-phase high-performance liquid chromatography. Proteins in each fraction were separated by two-dimensional gel electrophoresis. Then the gel was stained by Coomassie Brilliant Blue. Protein spots in which the expression levels were significantly different between cancer and normal control were identified by LC-MS/MS. One hundred and two spots were upregulated, and 84 spots were downregulated in serum samples obtained from patients with pancreatic cancers, and 58 proteins were identified by mass spectrometry. These candidate proteins were validated using western blot analysis and enzyme-linked immunosorbent assay (ELISA). As a result of these validation process, we could confirm that the serum levels of apolipoprotein A-IV, vitamin D-binding protein, plasma retinol- binding protein 4, and tetranectin were significantly decreased in patients with pancreatic cancer. 1. Introduction Pancreatic cancer is one of the most lethal malignancies, with a 5-year survival rate of only 4-5% [1]. The major reasons for the poor prognosis may be late diagnosis and limited therapeutic options; early diagnosis of pancreatic cancer is a pressing clinical problem. Serum levels of the conventional tumor markers includ- ing carcinoembryonic antigen (CEA) and the Lewis blood group carbohydrate antigen (CA19-9) often remain in nor- mal range at early stages of this malignancy [2]. Therefore, search for novel biomarkers of pancreatic cancer is needed. Recent advances in proteomic technologies have pro- vided promising ways to discover and identify novel bio- markers in various fields of clinical medicine. Although there has been long and uncertain path from marker discovery to clinical utility [3], sophisticated technologies have facilitated the discovery of potential tumor markers with improved sensitivities and specificities for the diagnosis of cancer patients [4]. Also, proteome analysis can lead to biomarkers that may be useful in the prediction of clinical response to anticancer therapy [5]. Surface enhanced laser desorption/ionization time-of- flight mass spectrometry (SELDI-TOF MS) is a represen- tative example of a proteomics technique for the high- throughput fingerprinting of serum proteins and peptides and biomarker discovery [6]. Using this technology, we could detect and identify novel diagnostic markers for alcohol abuse [7] and also a new prognostic marker for pancreatic cancer [8]. One of the technical challenges in serum proteome analy- sis is that serum contains thousands of proteins and peptides