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Article

Lumbar Cerebrospinal Fluid Proteome in Multiple Sclerosis: Characterization by Ultrafiltration, Liquid Chromatography, and Mass Spectrometry

Supporting Info

Jean-Paul Noben,^†^‡ Debora Dumont,^†^‡ Natalia Kwasnikowska,^§ Peter Verhaert, Veerle Somers,^† Raymond Hupperts, Piet Stinissen,^† and Johan Robben*^†

Biomedical Research Institute, Hasselt University, and School of Life Sciences, transnationale Universiteit Limburg, Diepenbeek, Belgium, Department of Mathematics-Physics-Informatics, Theoretical Computer Sciences, Hasselt University, Diepenbeek, Belgium, Faculty of Applied Sciences, Analytical Biotechnology Group, Delft University of Technology, Delft, The Netherlands, and Department of Neurology, University Hospital Maastricht, Maastricht, The Netherlands

J. Proteome Res., 2006, 5 (7), pp 1647–1657

DOI: 10.1021/pr0504788

Publication Date (Web): May 19, 2006

†

Biomedical Research Institute, Hasselt University, and School of Life Sciences, transnationale Universiteit Limburg.

‡

Both authors contributed equally to this work.

Department of Mathematics-Physics-Informatics, Hasselt University.

Delft University of Technology.

University Hospital Maastricht.

Corresponding author. Johan Robben, Biomedical Research Institute (BIOMED), Hasselt University, Agoralaan building A, 3590 Diepenbeek, Belgium. E-mail: johan.robben@uhasselt.be. Fax: +32−11−26 92 09.

Abstract

Neurological diseases, including multiple sclerosis (M.S.), often provoke changes in the functioning of the endothelial and epithelial brain barriers and give rise to disease-associated alterations of the cerebrospinal fluid (CSF) proteome. In the present study, pooled and ultrafiltered CSF of M.S. and non-M.S. patients was digested with trypsin and analyzed by off-line strong cation-exchange chromatography (SCX) coupled to on-line reversed-phase LC−ESI−MS/MS. In an alternative approach, the trypsin-treated subproteomes were analyzed directly by LC−ESI−MS/MS and gas-phase fractionation in the mass spectrometer. Taken together, both proteomic approaches in combination with a three-step evaluation process including the search engines Sequest and Mascot, and the validation software Scaffold, resulted in the identification of 148 proteins. Sixty proteins were identified in CSF for the first time by mass spectrometry. For validation purposes, the concentration of cystatin A was determined in individual CSF and serum samples of M.S. and non-M.S. patients using ELISA.

Keywords: centrifugal ultrafiltration • cerebrospinal fluid • cystatin A • gas-phase fractionation • liquid chromatography • mass spectrometry • multiple sclerosis • proteomics • validation

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