Quantitative Proteomics Analysis Of Cerebrospinal Fluid In Tuberculous Meningitis

Tuberculous meningitis (TBM), a kind of non-suppurative inflammation of the meninges, is caused by Mycobacterium tuberculosis. It can cause severe neurologic deficits and high mortality. Its prognosis largely depends on early diagnosis, as well as timely treatment.TBM is always misdiagnosed with purulent meningitis and cryptococcal meningitis for typical clinical manifestations.Proteins are essential parts of human organism and basically participate in every process of our body functions. A systematic research on protein groups, particularly proteomics, will directly reveal the changed mechanisms of life under physiological or pathological conditions.In recent years, the further exploration on differential proteomics and the gradual perfection of biological information technology have opened a new door for the research on the mechanism and potential biomarkers for diagnosis of the diseases.Central nervous system (CNS) diseases can cause changes in the protein expression of cerebrospinal fluid (CSF).Meanwhile, Information on the biology and pathology of CNS diseases is reflected by changes in the proteins and polypeptides of the CSF.So CSF can be used to search the target proteins in the pathogenesis and the biomarkers for early diagnosis. CSF was characterized of low protein level and many proteins were albumen and immune globulin.Conventional methods were difficult to separate and identify these proteins in CSF. At present, differential proteomics provides a powerful approach for screening for alterations in protein levels and posttranslational modifications associated with diseases.Many scholars have discovered potential biomarkers of diseases through proteomics methods.However, the global proteomic profiling of human TBM CSF has come about very slowly, and changes in the CSF proteomes of TBM patients are rarely reported.We first use fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) and Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) to explore differently expression of proteins profiles of CSF in TBM,screen key proteins related to the disease, and provide new clues to the diagnosis of TBM and research on mechanism. The CSF of acute TBM patients(20cases) and control people(20cases) were all collected from the shanghai public health clinic center between May2009and July2010.We isolated CSF proteins of TBM and control groups by2D-DIGE. Protein spots that exhibited statistical differences between TBM and control groups with>2.0difference were screened using DeCyder DIA V5.0software.The differential proteins were identified by MALDI-TOF-MS. The subcellur location and function of the proteins were elucidated by UniProt knowledgebase (Swiss-Prot/UniProtKB) and Gene Ontology(GO) Database.Some differential proteins was verified by enzyme-linked immunosorbent assays (ELISA).2D-DIGE patterns of TBM and control groups were established.13differently expressed proteins spots with Ratio>2.0were found by Gel-analysis software DeCyder5.0between TBM and control group. Through MALDI-TOF-MS and database retrieve,6differential proteins were identified,of which2proteins were up-regulated and4proteins were down-regulated in TBM compared with control groups.Up-regulated proteins were Apolipoprotein A-IV(APOA4) and Orosomucoid1(ORM1). Down-regulated proteins were Apolipoprotein E(APOE), Apolipoprotein A-I (APOA1),Vitamin D-binding protein(GC),Transthyretin(TTR).The differential expression of APOE in CSF was confirmed by ELISA.To further screen more key proteins associated to the disease, We then employed isobaric tags for relative and absolute quantitation labeling (iTRAQ) coupled with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS) to study CSF proteomes of TBM and compare the differential protein expressions of CSF among TBM,cryptococcal meningitis and control groups. After LC-MS/MS analysis,208proteins in TBM CSF were identified and quantified. According to the annotation from GO database, the most proteins identified locate in extracellular part, membrane,cytoplasm and cytoskeleton etc, and function as binding, catalytic activity, enzyme regulator activity, and transporter activity, and most might be involved in biological regulation,cellular process,response to stimulus,metabolic process,and so on A total of9differentially expressed proteins were screened compared with cryptococcal meningitis and control groups;6proteins were up-regulated and3were down-regulated. Up-regulated proteins were APOA4, Apolipoprotein B-100,(APOB), ORM1, a1-antichymotrypsin (SERPINA3), Cathelicidin antimicrobial peptide (CAMP), S100Calcium-binding Protein A8(S100A8). Down-regulated proteins were APOE, Calsyntenin1(CLSTN1), Prostaglandin D2synthase (PTGDS).S100A8and APOB were verified to be over-expressed in TBM CSF by ELISA.In summary, protein profiles of CSF in TBM were changed significantly.We identified and quantified12differential proteins by using2D-DIGE and iTRAQ coupled with MS. These proteins may be associated with TBM pathogenesis that restrain/accelerate disease occurrence and development and provides clues for further investigations on the molecular mechanism of TBM and its diagnostic biomarkers.Some proteins such as PTGDS, CLSTN1and S100A8are first time to be found abnormal expression in CSF of TBM and may be potential biomarker of TBM.Meanwhile,Our study demonstrated that differential protein profiles of CSF can be quickly found by2D-DIGE and iTRAQ-LC-MS,but the proteins identified by the two methods have cross and mutual complement. We must employe the two quantitative proteomics methods to comprehensively understand the CSF proteomes of TBM.