URINARY EXOSOMES AND PROTEOMICS Pyong-Gon Moon, 1,2 Sungyong You, 3 Jeong-Eun Lee, 1,2 Daehee Hwang, 3 and Moon-Chang Baek 1,2 * 1 Department of Molecular Medicine 2 Cell and Matrix Biology Research Institute, School of Medicine, Kyung- pook National University, Daegu 700-422, Republic of Korea 3 School of Interdisciplinary Bioscience and Bioengineering & Department of Chemical Eng., POSTECH, Pohang 790-784, Republic of Korea Received 21 October 2009; revised 23 July 2010; accepted 23 July 2010 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mas.20319 A number of highly abundant proteins in urine have been identi- fied through proteomics approaches, and some have been con- sidered as disease-biomarker candidates. These molecules might be clinically useful in diagnosis of various diseases. However, none has proven to be specifically indicative of perturbations of cellular processes in cells associated with urogenital diseases. Exosomes could be released into urine which flows through the kidney, ureter, bladder and urethra, with a process of filtration and reabsorption. Urinary exosomes have been recently sug- gested as alternative materials that offer new opportunities to identify useful biomarkers, because these exosomes secreted from epithelial cells lining the urinary track might reflect the cellular processes associated with the pathogenesis of diseases in their donor cells. Proteomic analysis of such urinary exosomes assists the search of urinary biomarkers reflecting pathogenesis of various diseases and also helps understanding the function of urinary exosomes in urinary systems. Thus, it has been recently suggested that urinary exosomes are one of the most valuable targets for biomarker development and to understand pathophy- siology of relevant diseases. # 2011 Wiley Periodicals, Inc. Mass Spec Rev Keywords: urinary exosomes; urogenital diseases; biomar- kers; proteomics I. INTRODUCTION The attempts to identify disease biomarkers, for diagnostics and prognostics, increase tremendously during the last 10 years as proteomics technology has been developed and opti- mized (Azad et al., 2006; Qian et al., 2006; Geho et al., 2007; Tao & Lazarev, 2007). Urine could be easily used for clinical as well as basic research because urine might be obtained in a simple, non-invasive, sufficient and stable fashion compared to other clinical samples (Theodorescu et al., 2006; Decramer et al., 2008; Caubet et al., 2010). Urine contains proteins, pep- tides, and metabolites derived from kidney as well as blood (Decramer et al., 2008). Therefore, urine is applicable to study with regards to urogenital diseases and non-urogenital diseases from associated organs in the body (Thongboonkerd, 2008). The advent of the proteomics era has enormously affected the methodology to study proteins, and has accelerated the ability to acquire information on proteins for basic and clinical research areas. The number of urine proteins identified during the past 5 years exceeded by 10-fold that of urine proteins found previously, and facilitated the search for urinary bio- markers (Spahr et al., 2001; Pieper et al., 2004; Adachi et al., 2006; Wang et al., 2006; Moon et al., 2008; Zurbig & Mischak, 2008). Various mass spectrometry techniques including 2-D PAGE-MS, surface-enhanced laser desorption/ionization time- of-flight mass spectrometry (SELDI-TOF MS), LC-MS/MS and capillary electrophoresis mass spectrometry (CE-MS), have been applied to analyze urine proteome. SELDI-TOF MS (Cadieux et al., 2004; Khurana et al., 2006; Poon, 2007), and CE-MS (Mischak et al., 2004; Weissinger et al., 2004; Haubitz et al., 2005; Wittke et al., 2005; Decramer et al., 2006) have been used for profiling approaches, and LC-MS/MS (Castagna et al., 2005; Sun et al., 2005; Adachi et al., 2006; Ru et al., 2006; Wang et al., 2006; Pieper, 2008; Smalley et al., 2008) and 2-D PAGE-MS (Thongboonkerd, Klein, & Arthur, 2003; Saito et al., 2005; Thongboonkerd, 2007) have been used for biomarker discovery. The advantages and disadvantages of each MS-based proteomics techniques have been reviewed in detail elsewhere (Fliser et al., 2007; Decramer et al., 2008). For these methods, usually the whole urine has been used. One of the challenges for biomarker identification is to reduce the dynamic range of protein concentrations in samples. To overcome this difficulty, an immunosubtraction method such as albumin depletion, affinity chromatography method such as lectin (Yang & Hancock, 2004, 2005; Wang et al., 2006; Moon et al., 2008) and immobilized metal-ion chromatog- raphy (IMAC) columns (Belew et al., 1987; Hoffert et al., 2006; Villen & Gygi, 2008), and ligand library beads method (Righetti et al., 2006) have been introduced, and the identification efficiency of low-abundance proteins has been improved. However, it is still difficult to select good bio- marker candidates for the next validation step among proteins with differentially expressed proteins. Urinary exosomes are microparticles identified recently in urine (Pisitkun, Shen, & Knepper, 2004). Proteomic research of these urinary exosomes has become one of the newer trends in the field of urine-biomarker discovery. Various mammalian cells such as reticulocytes (Pan & Johnstone, 1983; Johnstone et al., 1987), mast cells (Skokos et al., 2001a, 2003; Valadi Contract grant sponsor: Korean Ministry of Education, Science and Technology; Contract grant sponsor: Regional Technology Innovation Program of the Ministry of Knowledge Economy; Contract grant num- ber: RTI04-01-01. *Correspondence to: Moon-Chang Baek, Department of Molecular Medicine, School of Medicine, Kyungpook National University, 101 Dongin-dong 2 Ga, Jung-gu, Daegu 700-422, Republic of Korea. E-mail: mcbaek@knu.ac.kr Mass Spectrometry Reviews # 2011 by Wiley Periodicals, Inc.