Research Article Feasibility of electrodialysis as a fast and selective sample preparation method for the profiling of low-abundant peptides in biofluids Considerable interest exists in endogenous peptides as potential biomarkers, since they act as signaling molecules and are formed by degradation of proteins. A crucial step in the profiling of these peptides is the sample preparation, which aims to enrich the low- abundant peptides, while removing interfering matrix compounds. In a feasibility study we examined the suitability of electrodialysis (ED) for this purpose. A custom-made device was developed from the low-binding material Kel-F. It consisted of two compartments separated by a dialysis membrane, over which a voltage was applied. One compartment served as donor (containing the sample), while the smaller acceptor compartment collected the peptides. The procedure was optimized by investigating the effect of the applied voltage, ammonium acetate buffer concentration, and ED duration using model peptides. Optimum conditions were found at 300 V (150 V/cm), 25 mM ammonium acetate buffer (pH 3.8) containing 20% v/v DMSO, and 10 min, respectively. With these optimized parameters, recoveries for the model peptides were found to be 35–85% (average 64%). Additionally, ED was successfully applied to the challenging synovial fluid biological sample (due to its high viscosity). In a synovial fluid sample from a rheumatoid arthritis patient, 27 peptides originating from 12 proteins were identified, of which a considerable fraction was not identified before with other methods. This demonstrates the usefulness and complementary nature of combining ED with nanoLC- MS for biomarker discovery. These results indicate that ED is promising as a fast and selective sample preparation method for the profiling of endogenous peptides. Keywords: Electrodialysis / Endogenous peptides / NanoLC-MS / Peptidomics / Sample preparation DOI 10.1002/elps.200800500 1 Introduction Recently, there is considerable interest in the analysis of endogenous peptides, also called the peptidome or the low- molecular-mass proteome [1–3]. Endogenous peptides may provide good biomarkers as they play important roles in signaling and communication, for example as hormones, growth factors, or cytokines. Furthermore, they are indica- tors of protease activity. Examples of peptide biomarkers are collagen telopeptides (e.g. CTX-II) for osteoarthritis, osteo- calcin for osteoporosis, and Pro-GRP for small-cell lung cancer [4]. A crucial step in the profiling of peptides is the sample preparation, which aims to make the sample suitable for analysis by removing interfering matrix components, such as proteins, and by selectively enriching the compounds of interest. This is of crucial importance as peptides are only present in low concentrations. For sample preparation of peptides various techniques exist, examples of which are liquid–liquid extraction, SPE, precipitation, dialysis, and ultrafiltration [5]. In practice, the sample preparation tech- niques that are most frequently used for peptide profiling studies are ultrafiltration, SPE, and precipitation. Aristoteli et al. compared these preparation techniques for plasma with respect to the number of peptides detected and the Jurre J. Kamphorst 1,2 Ubbo R. Tjaden 1 Rob van der Heijden 1,2 Jeroen DeGroot 3 Jan van der Greef 1,2,3 Thomas Hankemeier 1,2 1 Division of Analytical Biosciences, Leiden/Amsterdam Center for Drug Research, Leiden University, Leiden, The Netherlands 2 Centre for Medical Systems Biology, Leiden University, Leiden, The Netherlands 3 TNO Quality of Life, Food and Chemical Risk Analysis, Zeist, The Netherlands Received July 31, 2008 Revised November 18, 2008 Accepted November 19, 2008 Abbreviations: BAMD, bovine adrenal medulla dodecapeptide; ED, electrodialysis; FA, formic acid; RF, radio frequency; SF, synovial fluid Correspondence: Jurre J. Kamphorst, Division of Analytical Biosciences, Leiden/Amsterdam Center for Drug Research, Leiden University, P. O. Box 9502, 2300 RA Leiden, The Nether- lands E-mail: j.kamphorst@chem.leidenuniv.nl Fax: 131-71-5274277 & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com Electrophoresis 2009, 30, 2284–2292 2284