Differential Proteomics Study On Serum In Patients With Multiple Sclerosis

Objective: Multiple sclerosis is an autoimmune inflammatory disease of the central nervous system(CNS)with white matter demyelinateion as the main pathobiology feature. Disease mechanisms in multiple sclerosis at the molecular level remain poorly understood .The diagnosis is based on case history , clinical symptoms, physical examination,magnetic resonance imaging and detection of oligimmunoglobulins in cerebrospinal fluid (CSF).at present, no reliable proteinaceous disease biomarkers are available in serum yet.With the Human Genome Project completed, it has been realized that Although genomics provide a strong basis for human in the gene activity and the relevance of the disease , but the expression of gene is complexed , the same one gene may play a completely different role in different conditions and in different periods of time.It is in such circumstances, post genome project have emerged, including functional genomics, proteomics, transcriptomics, and many other research areas.The proteome research became the focus of attention because of the large-scale, high-throughput analysis of the biological function of proteins play a direct role in biological function.Proteome derived from the concept of protein and genome of heterozygosity of the two words .Proteomics, the science of identifying the entire protein complement of a cell, tissue or fluid at a given point in time, can provide insight into the mechanisms of disease. Proteomics is to study proteins, large-scale, systematic study of protein structure and the characteristics, including the dynamic changes in intracellular protein composition, the expression level of the state and post-translational modification of protein and the interaction and links between the proteins.In our study,we used two-dimensional difference gel electrophoresis (2D DIGE) technique, in combination with matrix-assisted laser desorption ionization (MALDI) time-of-fight/time-of-flight (TOF/TOF) mass spectrometry (MALDI/TOF/TOF/MS), to analysis the proteins in serum samples from multiple sclerosis patients and neuromyelitis optica patients and normal people.To identify specific serum biomarkers in patients with multiple sclerosisMethods: 1.Materials:Serum from five patients with multiple sclerosis(clinical definite multiple sclerosis CDMS ;laboratory supported definite multiple sclerosis, ISDMS) ,five patients with neuromyelitis optica ,and five persons with healthy control group were stored at -80℃until analysis.2.Excluding high-abundance proteins ,cold acetone precipitation:Serum protein is composed of at least more than 10000 kinds of forms, and most exist at very low abundance.And the recognition of low abundance proteins is very important because many low abundance proteins often exercise a very important role,However, high abundance serum proteins , including albumin, immunoglobulin and transferrin etc, will cover low-abundance proteins.The key issue of the serum proteome is to remove high abundance serum proteins effectively .Application of high abundance removal kit to remove the serum albumin, immunoglobulin etc.In order to remove the salt all serum samples were precipitated with 100% ice-cold acetone in a 4:1 ratio of acetone to serum at -20℃.3.Protein assay:Bradford protein quantitative4.2D-DIGE:Three serum samples pooled with cy2 lablled as internal standard, The samples were loaded on a pH gradient strip for isoelectric focusing on an IPGphor(Amersham Biosciences-GE Healthcare). The serum protein separation by isoelectric point.After IEF, the strips were equilibrated in equilibration solution for two times.Then the IPG strips were loaded on 11% polyacrylamide gels in Ettan DALT Six system (Amersham Biosciences-GE Healthcare, Uppsala, Sweden) for electrophoresis at 1.5 W/gel overnight until the bromophenol blue front reached the bottom of the gels. Serum proteins separated by the different molecular weight.5.Image and statistic analysis:Fluorescent images were collected with a Typhoon9410 scanner . Matching, quantification and statistic analysis was carried out with DeCyder software. Find the statistically significant points, Use matrix-assisted laser desorption ionization time-of-fight / time-of-flight mass spectrometry (MALDI-TOF-TOF/MS) to identify the protein spots. Finally, the database searching to identify the corresponding proteins.Results: 1.This experiment received the two-dimensional difference gel electrophoresis profiles according to separate the serum proteins of MS and control group with two-dimensional difference gel electrophoresis (2D-DIGE) technique. 2.This experiment separated the serum proteins successfully with two-dimensional difference gel electrophoresis (2D-DIGE) technique,and 22 different protein were accessed. Fifteen kinds of protein were identified with matrix-assisted laser desorption ionization time-of-fight /time-of-flight mass spectrometry.They are alpha-1-acid glycoprotein 1 precursor ,alpha-1-antitrypsin precursor,Complement C3 precursor (Fragment),26S proteasome non-ATPase regulatory,apolipoprotein A-I precursor,clusterin isoform 1,HP protein, Ig mu heavy chain disease protein,immunoglobulin J chain,Ig mu chain C region,IGKV1-5 protein, Felic,187kD protein etc.Conclusion : Fifteen kinds of specific proteins related with multiple sclerosis were identified and the functions of these proteins and their relationship with multiple sclerosis was preliminary analysised .But it remains unknown whether these proteins are related to the cause and pathogenesis of multiple sclerosis.