|dc.description.abstract||The main focus of this study was to use mass spectrometry-based proteomics to study
protein abundance in cerebrospinal fluid (CSF) to reveal proteins that could serve as
biomarker candidates in multiple sclerosis (MScl).
By combining a CSF pooling strategy and label-free relative quantification we
discovered 65 proteins of differential abundance between MScl patients and controls.
A selection of 17 biomarker candidates was further subjected to two independent
verification steps: using stable isotope dimethyl labeling coupled to Accurate
Inclusion Mass Screening (dimethyl-AIMS) and Stable Isotope Dilution Selected
Reaction Monitoring (SID-SRM) for targeted quantification. The SID-SRM study
included a larger patient cohort of 125 cases and controls. To our knowledge, this is
the first report of a larger SRM verification study for biomarker candidates in MScl.
The most interesting results from the biomarker discovery and verification study were
the significantly decreased abundance of Apolipoprotein D, Cystatin C, Kallikrein-6
and Alpha-1-acid glycoprotein 1 in MScl patients compared to controls.
Furthermore, we performed a comprehensive characterization of the normal
CSF proteome. We identified 18, 807 peptide mapping to 1987 proteins by applying
immuno-affinity depletion and SDS-PAGE for enhanced proteome coverage. We
obtain a comprehensive set of reference proteins that could further be used for
investigations in MScl. The experiment gave us the opportunity to examine the size
distribution on the SDS-PAGE gel of a selection of biomarker candidate proteins in
normal CSF, with the aim to reveal potential protein variants (isoforms, truncation
products and proteolytic processed products). We hypothesized that the identification
of non-tryptic peptides could indicate truncation products of proteins in CSF. 13 and
nine non-tryptic peptides were identified for the biomarker candidates Cystatin C and
Secretogranin-1, respectively. This information had immediate utility for
investigation in MScl. Based on the observed spread in size distribution of biomarker
candidate proteins in normal CSF, we aimed to obtain quantitative information of
proteins present in both high and low mass fractions on the gel and further target these
proteins to obtain an abundance ratio between RRMS (patients) and OIND (controls),
and investigate if these ratios differed for the same protein. The most striking
observation was the opposite regulation level in CSF of Secretogranin-1.||en