RESEARCH ARTICLE Pre-analytical influence on the low molecular weight cerebrospinal fluid proteome Frode S. Berven 1 , Ann C. Kroksveen 1, 2 , Magnus Berle 1 , Tarja Rajalahti 3 , Kristian Flikka 4, 5, 6 , Reidar Arneberg 7, 8 , Kjell-Morten Myhr 3, 9, 10 , Christian Vedeler 3, 9 , Olav M. Kvalheim 11 and Rune J. Ulvik 1, 12 1 Institute of Medicine, University of Bergen, Bergen, Norway 2 Department of Molecular Biology, University of Bergen, Bergen, Norway 3 Department of Neurology, Haukeland University Hospital, Bergen, Norway 4 Computational Biology Unit, Bergen Centre for Computational Science, Bergen, Norway 5 Proteomics Unit at University of Bergen (PROBE), Bergen, Norway 6 Department of Informatics, University of Bergen, Bergen, Norway 7 Pattern Recognition Systems AS, Bergen, Norway 8 Center for Integrated Petroleum Research, University of Bergen, Bergen, Norway 9 Department of Clinical Medicine, University of Bergen, Bergen, Norway 10 The National Competence Centre for Multiple Sclerosis, Haukeland University Hospital, Bergen, Norway 11 Department of Chemistry, University of Bergen, Bergen, Norway 12 Laboratory of Clinical Biochemistry, Haukeland University Hospital, Bergen, Norway Cerebrospinal fluid (CSF) is a perfect source to search for new biomarkers to improve early diagnosis of neurological diseases. Standardization of pre-analytical handling of the sample is, however, important to obtain acceptable analytical quality. In the present study, MALDI-TOF MS was used to examine the influence of pre-analytical sample procedures on the low molecular weight (MW) CSF proteome. Different storage conditions like temperature and duration or the addition of as little as 0.2 mL blood/mL neat CSF caused significant changes in the mass spectra. The performance of different types of MW cut-off spin cartridges from different suppliers used to enrich the low MW CSF proteome showed great variance in cut-off accuracy, stability and repro- ducibility. The described analytical method achieved a polypeptide discriminating limit of ap- proximately 800 pM, two to three orders of magnitude lower than reported for plasma. Based on this study, we recommend that CSF is centrifuged immediately after sampling, prior to storage at –807C without addition of protease inhibitors. Guanidinium hydrochloride is preferred to break protein-protein interactions. A spin cartridge with cut-off limit above the intended analytical mass range is recommended. Our study contributes to the important task of developing stand- ardized pre-analytical protocols for the proteomic study of CSF. Received: February 6, 2007 Revised: March 28, 2007 Accepted: April 23, 2007 Keywords: Biomarker discovery / Cerebrospinal fluid / Mass spectra / Pre-analytical / Principal component analysis Proteomics Clin. Appl. 2007, 1, 699–711 699 1 Introduction The interest for biomarker discovery in body fluids like serum, plasma and cerebrospinal fluid (CSF) has grown rapidly over the last few years as the improvement of mass spectrometers and new fractionation techniques have given researchers the opportunity to continuously dig deeper into Correspondence: Dr. Frode S. Berven, Institute of Medicine, Uni- versity of Bergen, 5021, Bergen, Norway E-mail: Frode.Berven@biomed.uib.no Fax: 147-55586360 Abbreviations: CSF , cerebrospinal fluid; CTA, cellulose triacetate; MWCO, molecular weight cut-off; PCA, principal component analysis; PES, poly(ethersulphone) DOI 10.1002/prca.200700126  2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clinical.proteomics-journal.com