RELAXIN AND RELATED PEPTIDES: FIFTH INTERNATIONAL CONFERENCE Evaluation of Relaxin’s Antifibrotic Action by SELDI-TOF Mass Spectrometry-Based Profiling of Relaxin Knockout Mice, a Model of Progressive Fibrosis Eleni Giannakis, a Mary Macris, a Linda Chan, a Geoffrey W. Tregear, a,b Chrishan S. Samuel, a,b and John D. Wade a,c a Howard Florey Institute, b Department of Biochemistry and Molecular Biology, and c School of Chemistry, University of Melbourne, Victoria 3010, Australia Surface-enhanced laser desorption ionization–time-of-flight mass spectrometry was employed to identify potential biomarkers for early-onset fibrosis. These biomarkers were then used to evaluate the efficacy of relaxin to reverse or ameliorate the develop- ment of the condition. Key words: fibrosis; protein profiling; relaxin knockout mice; SELDI-TOF mass spectrometry Introduction Fibrosis (organ scarring) is a leading cause of morbidity and mortality. It is character- ized by an overproduction of collagen and causes a hardening of vital tissues, leading to a progressive loss of their function. 1 The pep- tide hormone relaxin (RLX) has known an- tifibrotic properties: it reduces collagen syn- thesis and promotes collagen degradation via its ability to upregulate matrix-degrading pro- teases (matrix metalloproteases). To be most effective, however, RLX needs to be admin- istered in the early stages of disease. 2 In or- der to develop diagnostics for the onset of fi- brosis to maximize the therapeutic potential of RLX and provide a means to measure the effi- cacy of therapeutic intervention strategies, we used a mouse model of age-related fibrosis and surface-enhanced laser desorption ionization– time-of-flight mass spectrometry (SELDI-TOF Address for correspondence: Professor John D. Wade, Howard Florey Institute, University of Melbourne, Victoria 3010, Australia. Voice: +61 3 8344 7285; fax: +61 3 9348 1707. john.wade@florey.edu.au MS; Bio-Rad, Inc., Hercules, CA, USA) 3 to identify potential biomarkers for early-onset fibrosis. Methods Samples The RLX-1 knockout (RLX KO) mouse, which exhibits age-related fibrosis progression, was used as the model for this study. RLX KO mice were administered recombinant RLX in citrate buffer, which reverses the diseased phenotype, or as a control citrate buffer (vehicle) alone. Sera from healthy and diseased animals were collected at differ- ent time points: 9 months (early onset of fibrosis) and 12 months (established fibro- sis). Serum was diluted in 9 M urea, 1% 3-[(3-cholamidopropyl)-dimethylammonio]-1- propanesulfonate (CHAPS), phosphate- buffered saline (PBS; 1:2) and incubated for 30 min on ice. Samples were then diluted in PBS (1:30) in preparation for analysis via SELDI-TOF MS. Relaxin and Related Peptides: Fifth International Conference: Ann. N.Y. Acad. Sci. 1160: 350–352 (2009). doi: 10.1111/j.1749-6632.2009.03957.x C  2009 New York Academy of Sciences. 350