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

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