Abstract
Two-dimensional difference gel electrophoresis (2D-DIGE) has been used for identification of possible biomarkers in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients. However, in different studies inconsistent results have been obtained. We wanted to analyze the diagnostic value of 2D-DIGE in early MS patients by comparing protein patterns between single and pooled samples of MS patients and controls. CSF samples of 20 MS patients and 10 control subjects were processed with 2D-DIGE. The so obtained protein patterns were analyzed with DeCyder 6.5 software, whereby we described variation of patterns presented in one gel as well as between different gels. Even when running single samples of patients of the same group in one gel, variation of protein patterns was high. The number of identified spots with different protein level varied between 4 and 30, depending on which sample batches were compared. We did not find a consistent pattern throughout all possible batch combinations. The inter-individual variation of protein expression as well as the susceptibility of 2D-DIGE for methodological variations makes use of 2D-DIGE as a diagnostic tool for MS and for detection of possible candidate biomarkers difficult, since detected proteins vary depending on which samples are compared.
Highlights
Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system with vast heterogeneity between individual patients and different immunologic processes involved [1].The exact immunologic mechanisms leading to demyelination are still poorly understood and various approaches to find candidate genes, proteins or cells were of little success so far
In contrast to previous studies, we did not aim to identify single proteins by spot-picking, but we focused on protein patterns performing analyses with a statistical software in order to describe the accordance of these patterns throughout all samples and to analyze the consistency in obtaining specific protein spots
We aimed to analyze limitations of 2DE or two-dimensional difference gel electrophoresis (2D-DIGE) in proteomic studies of the cerebrospinal fluid (CSF) in order to explore possible reasons why results of previous studies in MS patients have been so controversial [5,6,7,8,9,10,11,12,13]. It seems obvious using 2D-DIGE for identification of proteins in the CSF of MS patients. It is a high-resolution method allowing protein separation by molecular weight and charge with consequent spot picking from the gels
Summary
Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system with vast heterogeneity between individual patients and different immunologic processes involved [1].The exact immunologic mechanisms leading to demyelination are still poorly understood and various approaches to find candidate genes, proteins or cells were of little success so far. 2D-DIGE provides the advantage of performing analyses on a reduced number of gels with comparison of samples in the same gel run under the same conditions and with a consistent pool-standard throughout all gels [14,15,16]. Results of these approaches were inconsistent so far, and the results of previous experiments could not be confirmed by later repetition of different
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