The Prostate 61:171^181 (2004) AComprehensive Characterization of the Peptide and Protein Constituents of Human Seminal Fluid Kim Y.C. Fung, 1 L. Michael Glode, 2 Spencer Green, 2 and Mark W. Duncan 1 * 1 Biochemical Mass Spectrometry Facility,University of Colorado Health Sciences Center, Denver,Colorado 2 Medical Oncology, School of Medicine,University of Colorado Health Sciences Center, Denver,Colorado BACKGROUND. Knowledge of the peptide and protein components of seminal fluid and their role in prostate diseases including benign prostatic hyperplasia and prostate carcinoma is scant. We have undertaken a proteomic analysis of semen as a forerunner to identifying sensitive and specific diagnostic markers of prostatic diseases; to aid in improved therapeutic intervention; and, to enhance our understanding of prostate health and disease. METHODS. Peptide and protein components of pooled human seminal fluid (n ¼ 5) were separated by gel electrophoresis (1D and 2D) and identified by either matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) or capillary liquid chromatography tandem mass spectrometry (LC–MS/MS). RESULTS. Analysis by two-dimensional electrophoresis (2DE) established that there were multiple post-translational variants of the majority of the proteins. Hormones, growth factors and bioactive peptides were detected and identified. CONCLUSIONS. We have identified over 100 protein and peptide components of normal human seminal fluid. Prostate 61: 171 – 181, 2004. # 2004 Wiley-Liss, Inc. KEY WORDS: peptides; proteins; proteomics; prostate cancer; biomarkers; PSA INTRODUCTION The incidence of prostate-related diseases such as prostatitis, benign prostatic hyperplasia, and prostate carcinoma is rapidly increasing in the developed world. In American men, prostate cancer is the most common cancer, excluding skin cancer, and is the second leading cause of cancer deaths, exceeded only by lung cancer [1]. It was estimated that in 2002, 189,000 men in the US would be diagnosed with prostate cancer and that over 30,000 deaths would be attributable to this disease [2]. Although the etiology of prostate cancer is unknown, the risk of developing the disease increases with age and is influenced by race, genetic disposition and environmental factors such as diet and lifestyle [3,4]. Decreasing the mortality associated with prostate cancer relies on increased public awareness, earlier diagnosis, and improved therapeutic strategies [5]. Consequently, specific, sensitive, simple, cost effec- tive, and rapid clinical tests for prostate cancer are of considerable importance. Currently, the primary biomarker for prostate cancer is prostate-specific antigen (PSA), a 33-kDa protein produced and secreted principally by the prostate epithelium and the epithelial lining of the periurethral glands. Since PSA testing was first introduced into clinical practice in the late 1980s, analysis has facilitated early diagnosis of prostate cancer. The relationship between serum PSA levels and prostate cancer risk has been well documented, and in part because of PSA testing, mortality from prostate cancer has decreased significantly over the last decade [6]. For instance, a man with a serum PSA level of 4 ng/ml has approximately a 20 – 30% risk of having prostate cancer [7]; for PSA levels greater than 10 ng/ml the risk of prostate cancer rises to between 42 and 64% [8,9]. Nevertheless, a single PSA determination is less *Correspondence to: Mark W. Duncan, Department of Pediatrics, Division of Endocrinology, UCHSC, 4200 East 9th Avenue, B151, Denver, CO, 80262. E-mail: mark.duncan@uchsc.edu Received 12 November 2003; Accepted 12 January 2004 DOI 10.1002/pros.20089 Published online 24 March 2004 in Wiley InterScience (www.interscience.wiley.com). ß 2004 Wiley-Liss, Inc.