Screening And Identifying Of Serum Biomarkers For Pulmonary Tuberculosis And The Traditional Chinese Medicine Syndrome By Proteomics Technology

Background:Tuberculosis (TB) is a chronic respiratory infectious disease caused by Mycobacterium tuberculosis (MTB). Despite the availability of an effective therapy, pulmonary TB (PTB) continues to be a major global public health problem. Especially in developing countries, the epidemic situation of TB is alleviated slowly. The reversal of this scenario will require the development of new strategies to increase the quality and speed of TB diagnosis. Biomarkers play an irreplaceable role in early diagnosis, disease surveillance, efficacy and prognostic evaluation of the disease. At the present stage, there are few effective biomarkers for early diagnosis of TB. Therefore, how to use new technology and means to discover and verify more sensitive and specific biomarkers for early diagnosis of TB is a major challenge and urgent task for the disease control. The emergence of proteomics technology makes the analysis of all the proteins in the serum possible. The surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF MS), matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and linear ion chromatograph mass spectrometry/mass spectrometry (LC-MS/MS) as powerful proteomics technology can be used in detection effective biomarkers for TB.Methods:SELDI-TOF MS combined with Weak cation exchange (WCX) magnetic beads was used to screen serum samples from180cases of pulmonary TB and211control subjects. A classification model was established by Biomarker Pattern Software (BPS). Candidate protein biomarkers were purified by reverse phase-high performance liquid chromatography (RP-HPLC), identified by MALDI-TOF MS, LC-MS/MS and validated using enzyme-linked immunosorbent assay (ELISA).Results:The protein profile from the240serum samples of the training set (consisting of120cases of TB patients,20cases of lung cancer patients,20cases of pneumonia patients,20cases of chronic obstructive pulmonary disease (COPD) patients and60cases of healthy volunteers) were analyzed with Biomarker Wizard software,35protein peaks were found to discriminate patients with TB and non-TB control subjects (P<0.01, Fold>1.5),69protein peaks were found to discriminate patients with TB and the differential diseases group (P<0.01, Fold≥1.5). The diagnosis model I based on the four biomarkers (2554.6,4824.4,5325.7, and8606.8m/z) was established which could distinguish TB from the non-TB controls with the sensitivity of83.3%and the specificity of84.2%. The diagnosis model Ⅱ based on the three biomarkers (4180.1,5325.7, and15103.6m/z) was established which could distinguish TB from the differential diseases controls with the sensitivity of87.5%and the specificity of90.0%. The candidate biomarker with m/z of2554.6was found to be up-regulated in TB patients. It was identified as a fragment of fibrinogen, alpha polypeptide isoform alpha-E preproprotein by RP-HPLC and LC-MS/MS. Analysis in patients with TB using ELISA showed increased fibrinogen degradation product (FDP)(5,005±1,297vs.4,010±1,181ng/mL, P<0.05) and in142patients showed elevated plasma fibrinogen levels.Conclusions:(1) SELDI-TOF MS combined with WCX magnetic beads can detect the protein peaks with good effectiveness and reproducibility by optimizing the experimental conditions.(2) Discriminating protein peaks were detected and a diagnostic model for TB with high sensitivity and specificity was developed using mass spectrometry combined with magnetic beads. This provided a biological basis for rapid diagnosis of TB using serum proteomics technology.(3) Fibrinogen was identified as a potential biomarker for TB and showed diagnostic values in clinical application. This provided a new foundation for clarifying TB pathogenesis and improving the standards of efficacy evaluation. Background:Chemotherapy is the mainstay of modern pulmonary TB control, while body injure, multidrug-resistant TB may be produced. Traditional Chinese medicine (TCM) can enhance apparently the effect of anti-tuberculosis drug, promote the absorption of the foci in the lung and reduce the toxicity of drug in chemotherapy. The effect of the combined treatment of TCM and Western Medicine is probably relevant with the strengthening of body immunity. In TCM, the determinations of treatment based in pathogenesis obtained through differentiation of symptoms and signs of TB. Determinations of treatment based in pathogenesis obtained through differentiation of symptoms and signs means deducing the causes of a disease and nature of disease according to the outer signs of a disease. Syndrome proteomics guided by the theory of syndrome, is applying the method of the formation of syndrome and to interpret the nature of syndrome in the level of integer proteins expression. So, it will help to reveal the biochemistry basis and pathogenesis of syndromes in TB.Methods:One hundred and eighty patients with TB were typed by syndrome differentiation and brought into71cases of FYX syndrome,64cases of YXHW syndrome and45cases of QYLX syndrome. SELDI-TOF MS combined with WCX magnetic beads was used to screen serum samples. A classification model was established by BPS. Candidate protein biomarkers were purified by RP-HPLC, identified by MALDI-TOF MS, LC-MS/MS and validated by ProteinChip Immunoassays.Results:The protein profile from the120serum samples of the TB patients (consisting of50cases of FYX syndrome,40cases of YXHW syndrome and30cases of QYLX syndrome) were analyzed with Biomarker Wizard software. Analysis of the common of changes in syndromes, we found12protein peaks to discriminate FYX syndrome and the other two syndromes (P<0.001),12protein peaks to discriminate YXHW syndrome and the other two syndromes (P<0.001) and34protein peaks to discriminate QYLX syndrome and the other two syndromes (P<0.001). Seven protein peaks were found to be down-regulated gradually from the syndrome of FYX to YXHW to QYLX (P<0.001). The diagnosis model for TB syndrome based on the five biomarkers (3961.7,4679.7,5646.4,8891.2and9416.7m/z) was established which could detect74.0%,72.5%and96.7%for classifying FYX syndrome patients, YXHW syndrome patients, QYLX syndrome patients. The candidate biomarker with m/z of9416.7was identified as a fragment of apolipoprotein C-III and validated by ProteinChip Immunoassays.Conclusions:(1) The differential protein peak of serum protein spectrum in TB patients with different syndrome may be internal material basis of external manifestation of syndrome, and close relationships among FYX, YXHW and QYLX syndrome, which are considered three different developing phases of TB. It is hopeful to reveal the nature of TCM by identifying the differential protein.(2) A diagnostic model for TB syndrome was developed using mass spectrometry combined with magnetic beads. This provided a biological basis for the determinations of treatment based different TB syndromes.(3) Apolipoprotein C-III was identified as a potential biomarker for TB syndrome and showed diagnostic values in clinical application.