The Research Of Separating, Identifying Low Molecular Marker Proteins From Pulmonary Tuberculosis Serum

Tuberculosis ( TB ) is a chronic respiratory infectious disease caused by the pathogen Mycobacterium tuberculosis (MTB).TB remains an important global public health problem, because about one-third of the world's populations are infected with the MTB and there are 10 million new cases and 3 million deaths annually. However, the technology diagnosis of tuberculosis is not ideal. It is very important to find a convenient, fast, high sensibility and specificity diagnosis method to control the prevalence and spread of tuberculosis.In recent years,Surface-enhanced laser desorption ionization time-of-flight mass spectrometry is developing as a new technique to identification and screening serum trace proteins in proteomics research.With the advantage of high-throughput and high sensitivity it has been used for variety of tumor markers detection research. We have screened outed 6 most important diognosing biomarkers ( 4953.58Da, 4748.19Da,4895.39Da, 4480.15Da, 6933.89Da,8778.35Da) from pulmonary tuberculosis serum.The accurate rate was 98.33%, sensitivity 96.67%, specificity 100%, positive predictive value 100%,negative predictive value 96.77%, when this 6 proteins were used to establish a diagnosis model. The deficiency of SELDI is that it only give molecular weight information of the proteins but not the sequences.Thus, the purpose of this study is to separate and identify these protein markers of pulmonary tuberculosis.Objective: 1.To optimize the resolution and sensitivity of the Low-molecular-weight proteins (<10kD) on the the one-dimensional SDS-PAGE by means of improving sample handling, electrophoresis conditions and staining methods etc,so they can be identificated by Mass Spectrum. 2.To separate, purify and identify the protein markers the most diagnostic value of TB by 1D-PAGE-LC-MS/MS, the property of the proteins were analysised by bioinformatics.Method:1. The samples were treated with isoelectric precipitation , acetonitrile precipitation and Albumin/IgG Removal Kit respectively ,then surveyed the status of high abundance proteins removal and aim protein residue by electroph oresis, Determination of protein concentration.2.The SDS-PAGE,Tricine-SDS-PAGE,4%-12% Bis-tris NuPAGE electrophoresis systemses were use to resolve the proteins less than 10 kD, and the electrophoresis conditions, including the separation gel was added urea or glycerol, the electro phoresis voltage, the staining methods were optimized.3.Tuberculosis-related protein biomarkers had been discovered ,separated and identified by 1D electrophoresis, sliced and enzyme, HPLC-ESI-MS/MS and Bioinformatics analysis.Results1.The ACN extracts showed that approximately 95% of all proteins are removed from the serum, both isoelectric precipitation and Albumin/IgG Removal Kit can move most multiple high-abundance proteins enabled the detection of the low abundance serum protein and the latter can specificly remove most albumin and IgG which constitute about 75% of the total serum.2.The target proteins from the band were separated and identified by sliced and enzyme, HPLC-ESI-MS/MS and Bioinformatics analysis. The TB protein maker is likely to be Apolipoprotein A-II,for the sequence coverage of 21%, three non-repeat peptides have been identified,this results more reliable.3.The results from bioinformatics analysis showed Apoli poprotein Aâ…¡is a secreted protein, it contains an amino acid chain of 77 (or 76) amino acid, MW = 8 707.9Da (or MW = 8579.78), pI = 5.05, may be the same as the amino acid sequences of the protein 8778 Da. The occurrence,development of tuberculosis closely related to lipid metabolism, ApoA-II also inextricably linked with lipid metabolism, So ApoAII may be TB's specific marker.Conclusion:1.Acetonitrile precipitation method can remove the vast majority of high abundance serum proteins at the same time gain more small molecular weight 15kD protein,is suitable for separation of serum proteins below 15kD.2.Tricine - SDS-PAGE has advantages in separation of small molecules below 20kD.The conventional Coomassie staining simple, convenient, compatibility with mass spectrometry identification.3.The protein maker was successfully separated and identified by Tricine - SDS-PAGE, sliced and enzyme, HPLC-ESI-MS/MS and Bioinformatics analysis it suggests that this technology roadmap can be used for the isolation and identification of small molecules in serum .4.Apo AII is likely to be tuberculosis-related serum marker.