基于蛋白组学的肺结核病及其中医证候血清标志物筛选与鉴定
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摘要
研究背景：结核病(Tuberculosis, TB)是由结核分枝杆菌(Mycobacterium tuberculosis, MTB)引起的一种慢性传染病。结核病疫情下降缓慢,肺结核(pulmonary tuberculosis, PTB)仍是影响全球特别是发展中国家公共卫生的重大传染疾病,急需新的策略提高肺结核的早期诊断质量。生物标志物对于疾病的筛查、诊断、预后、疗效判断以及检测十分重要,因此,寻找更为灵敏的特征性的生物标志物是早期诊断肺结核的当务之急。蛋白组学技术的发展为从整体水平分析血清蛋白质提供了技术平台,表面加强激光解析电离飞行时间质谱(surface-enhanced laser desorption ionization time of flight mass spectrometry, SELDI-TOF-MS)结合基质辅助激光解析电离飞行时间质谱(matrix assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF MS)、线性液质联用离子解串联质谱技术(linear ion chromatograph mass spectrometry/mass spectrometry, LC-MS/MS)技术,作为全新的蛋白组学技术可实现对血清中特异性标志物的筛选和鉴定。
研究方法：采用弱阳离子(weak cation exchange, WCX)磁珠结合SELDI-TOFMS技术建立180例初治继发性肺结核患者和211例对照的血清蛋白指纹图谱,应用Biomarker Pattern Software (BPS)软件建立肺结核的诊断模型。通过反相高效液相色谱(reverse phase-high performance liquid chromatography, RP-HPLC)分离及纯化潜在的生物标志物蛋白,应用MALDI-TOF MS跟踪目标蛋白,进一步用线性液质联用离子肼串联质谱(linear ion chromatograph mass spectrometry/mass spectrometry, LC-MS/MS)进行分析,以鉴定潜在诊断标志物蛋白,并用酶联免疫吸附测定(enzyme-linked immunosorbent assay, ELISA)进行验证。
研究结果：120例肺结核和120例非肺结核(60例健康对照、20例肺癌、20例肺炎、20例慢性阻塞性肺病(chronic obstructive pulmonary disease, COPD))血清蛋白图谱比较,35个蛋白峰在肺结核和非肺结核组之间有显著差异(P<0.01,Fold≥1.5)。筛选2554.6,4824.4,5325.7和8606.8m/z四个蛋白峰构建了诊断模型Ⅰ,灵敏度为83.3%,特异度为84.2%,该模型显示在肺结核临床大规模的筛查具有较好的诊断能力,盲法检验灵敏度为75.0%,特异度为83.5%。以120例肺结核患者和60例肺结核其他疾病(肺癌、肺炎、COPD)筛选的差异峰为基础构建了由4180.1,5325.7,15103.6m/z3个蛋白峰组成的诊断模型Ⅱ,灵敏度为87.5%,特异度为90.0%。该模型在临床上对于有相似症状以及相似影像学改变的肺结核和其他肺部疾病的鉴别具有更强的应用性,该模型盲法灵敏度为81.7%,特异度为85.0%,阳性预测值为84.5%。在肺结核中相对高表达的2554.6m/z蛋白峰经RP-HPLC分离纯化,并利用MALDI-TOF MS及LC-MS/MS进行鉴定,为纤维蛋白原(fibrinogen, alpha polypeptide isoform alpha-E preproprotein)的一个片段。血清ELISA实验结果证实肺结核组的纤维蛋白原降解产物(fibrinogen degradation product, FDP)量均值高于健康对照组(5,005±1,297vs.4,010±1,181ng/mL,P＜0.05),142例临床随机收集的肺结核病人血浆中的纤维蛋白原含量(5.45±1.65g/L)要高于正常值范围(2.0-4.0g/L)。
结论：(1)建立纳米磁珠结合SELDI-TOF MS的肺结核蛋白指纹图谱检测技术,可快速稳定灵敏地构建肺结核蛋白指纹图谱；建立了RP-HPLC联合MALDI-TOF MS、LC-MS/MS鉴定SELDI-TOF MS特异差异峰技术。(2)筛选了肺结核相关的差异蛋白峰,构建了肺结核早期诊断的蛋白诊断模型,为建立肺结核病临床早期快速特异诊断的血清蛋白质组学标准,提高肺结核病的防治水平奠定基础。(3)鉴定和验证了纤维蛋白原可能为肺结核早期诊断的潜在生物标志物之一,为临床肺结核早期诊断奠定了基础。
研究背景：肺结核属于中医学“肺痨”范畴,目前用于肺结核治疗的化学药物虽有疗效但毒副作用大且易形成耐药性,而中医基于患者机体正虚和痨虫感染辨证施治,尤其是近年来中西医结合治疗在耐药肺结核和疑难重症肺结核治疗中取得了一定的疗效。证候是中医辨证施治的核心,肺结核中医证候的诊断尚无科学量化的标准,严重影响临床治疗效果。蛋白质组学技术的很多特征如整体性、动态性、时空性、复杂性等与中医证候的特点十分吻合,以蛋白质组为切入点,深入进行证候实质的研究,有利于从微观的角度动态地了解证候的物质基础,可为中医证候诊断分型的客观化提供依据。
研究方法：采用中医独立四诊辨证结合舌诊仪脉诊仪对180例肺结核患者进行辨证分型,纳入肺阴虚证(FYX)71例,阴虚火旺证(YXHW)64例,气阴两虚证(QYLX)45例。采用WCX磁珠结合SELDI-TOF MS技术建立血清蛋白指纹图谱,应用Biomarker Wizard软件比较各证候间的差异峰并进行证候共性分析。应用BPS软件建立肺结核证候的辨证模型。通过RP-HPLC分离及纯化潜在的生物标志物蛋白,应用MALDI-TOF MS和LC-MS/MS进行分析,以鉴定潜在证候标志物蛋白,并用蛋白芯片技术进行验证。
研究结果：50例FYX证、40例YXHW证、30例QYLX证血清蛋白指纹图谱两两比较并作共性分析,FYX证与与其他两证候比较均存在差异的蛋白峰有12个(P<0.001), YXHW证与其他两证候比较均存在差异的蛋白峰有12个(P<0.001),QYLX证与其他两证候比较均存在差异的蛋白峰有34个(P<0.001)；有7个蛋白峰在三证间的含量呈肺阴虚→阴虚火旺→气阴两虚逐渐降低变化。以三证候间两两有差异的74个差异峰(P<0.001)为基础构建了由3961.7、4679.7、5646.4、8891.2、9416.7m/z5个蛋白标志峰组成的肺结核中医证候蛋白质指纹图谱辨证模型,对于FYX证辨证的准确率为74.0%,对于YXHW证的准确率为72.5%,对于QYLX的准确率为96.7%。该模型对于肺结核三证候的分型具有较好的鉴别能力,盲法验证对于FYX证辨证的准确率为76.2%,对于YXHW证的准确率为70.8%,对于QYLX的准确率为80.0%。对筛选到的蛋白差异峰进行了鉴定和抗体芯片的验证,结果表明9416.7m/z蛋白峰是apolipoprotein C-Ⅲ载脂蛋白C-Ⅲ中的一段多肽。
结论：(1)筛选归纳了肺结核各证候特有的差异蛋白峰,可能是证候外在表现的内在病理机制和物质基础,为整体性评价肺结核中医证候的实质提供证据；归纳了7个蛋白峰在三证间的含量呈肺阴虚→阴虚火旺→气阴两虚逐渐降低变化,结果与中医理论关于肺结核证候病情演变相契合,差异的标志蛋白峰体现了肺结核证候间一定的相互关系及演变规律。(2)构建了肺结核中医证候蛋白辨证模型,为肺结核证候诊断的客观化提供蛋白质标准,有助于规范肺结核中医治疗的辨证施治。(3)鉴定和验证了载脂蛋白C-Ⅲ在肺结核三证候中差异表达,可能为肺结核中医证候分型的血清潜在标志物之一,可在临床和功能方面作进一步的验证。
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.
研究方法：采用弱阳离子(weak cation exchange, WCX)磁珠结合SELDI-TOFMS技术建立180例初治继发性肺结核患者和211例对照的血清蛋白指纹图谱,应用Biomarker Pattern Software (BPS)软件建立肺结核的诊断模型。通过反相高效液相色谱(reverse phase-high performance liquid chromatography, RP-HPLC)分离及纯化潜在的生物标志物蛋白,应用MALDI-TOF MS跟踪目标蛋白,进一步用线性液质联用离子肼串联质谱(linear ion chromatograph mass spectrometry/mass spectrometry, LC-MS/MS)进行分析,以鉴定潜在诊断标志物蛋白,并用酶联免疫吸附测定(enzyme-linked immunosorbent assay, ELISA)进行验证。
研究结果：120例肺结核和120例非肺结核(60例健康对照、20例肺癌、20例肺炎、20例慢性阻塞性肺病(chronic obstructive pulmonary disease, COPD))血清蛋白图谱比较,35个蛋白峰在肺结核和非肺结核组之间有显著差异(P<0.01,Fold≥1.5)。筛选2554.6,4824.4,5325.7和8606.8m/z四个蛋白峰构建了诊断模型Ⅰ,灵敏度为83.3%,特异度为84.2%,该模型显示在肺结核临床大规模的筛查具有较好的诊断能力,盲法检验灵敏度为75.0%,特异度为83.5%。以120例肺结核患者和60例肺结核其他疾病(肺癌、肺炎、COPD)筛选的差异峰为基础构建了由4180.1,5325.7,15103.6m/z3个蛋白峰组成的诊断模型Ⅱ,灵敏度为87.5%,特异度为90.0%。该模型在临床上对于有相似症状以及相似影像学改变的肺结核和其他肺部疾病的鉴别具有更强的应用性,该模型盲法灵敏度为81.7%,特异度为85.0%,阳性预测值为84.5%。在肺结核中相对高表达的2554.6m/z蛋白峰经RP-HPLC分离纯化,并利用MALDI-TOF MS及LC-MS/MS进行鉴定,为纤维蛋白原(fibrinogen, alpha polypeptide isoform alpha-E preproprotein)的一个片段。血清ELISA实验结果证实肺结核组的纤维蛋白原降解产物(fibrinogen degradation product, FDP)量均值高于健康对照组(5,005±1,297vs.4,010±1,181ng/mL,P＜0.05),142例临床随机收集的肺结核病人血浆中的纤维蛋白原含量(5.45±1.65g/L)要高于正常值范围(2.0-4.0g/L)。
结论：(1)建立纳米磁珠结合SELDI-TOF MS的肺结核蛋白指纹图谱检测技术,可快速稳定灵敏地构建肺结核蛋白指纹图谱；建立了RP-HPLC联合MALDI-TOF MS、LC-MS/MS鉴定SELDI-TOF MS特异差异峰技术。(2)筛选了肺结核相关的差异蛋白峰,构建了肺结核早期诊断的蛋白诊断模型,为建立肺结核病临床早期快速特异诊断的血清蛋白质组学标准,提高肺结核病的防治水平奠定基础。(3)鉴定和验证了纤维蛋白原可能为肺结核早期诊断的潜在生物标志物之一,为临床肺结核早期诊断奠定了基础。
研究背景：肺结核属于中医学“肺痨”范畴,目前用于肺结核治疗的化学药物虽有疗效但毒副作用大且易形成耐药性,而中医基于患者机体正虚和痨虫感染辨证施治,尤其是近年来中西医结合治疗在耐药肺结核和疑难重症肺结核治疗中取得了一定的疗效。证候是中医辨证施治的核心,肺结核中医证候的诊断尚无科学量化的标准,严重影响临床治疗效果。蛋白质组学技术的很多特征如整体性、动态性、时空性、复杂性等与中医证候的特点十分吻合,以蛋白质组为切入点,深入进行证候实质的研究,有利于从微观的角度动态地了解证候的物质基础,可为中医证候诊断分型的客观化提供依据。
研究方法：采用中医独立四诊辨证结合舌诊仪脉诊仪对180例肺结核患者进行辨证分型,纳入肺阴虚证(FYX)71例,阴虚火旺证(YXHW)64例,气阴两虚证(QYLX)45例。采用WCX磁珠结合SELDI-TOF MS技术建立血清蛋白指纹图谱,应用Biomarker Wizard软件比较各证候间的差异峰并进行证候共性分析。应用BPS软件建立肺结核证候的辨证模型。通过RP-HPLC分离及纯化潜在的生物标志物蛋白,应用MALDI-TOF MS和LC-MS/MS进行分析,以鉴定潜在证候标志物蛋白,并用蛋白芯片技术进行验证。
研究结果：50例FYX证、40例YXHW证、30例QYLX证血清蛋白指纹图谱两两比较并作共性分析,FYX证与与其他两证候比较均存在差异的蛋白峰有12个(P<0.001), YXHW证与其他两证候比较均存在差异的蛋白峰有12个(P<0.001),QYLX证与其他两证候比较均存在差异的蛋白峰有34个(P<0.001)；有7个蛋白峰在三证间的含量呈肺阴虚→阴虚火旺→气阴两虚逐渐降低变化。以三证候间两两有差异的74个差异峰(P<0.001)为基础构建了由3961.7、4679.7、5646.4、8891.2、9416.7m/z5个蛋白标志峰组成的肺结核中医证候蛋白质指纹图谱辨证模型,对于FYX证辨证的准确率为74.0%,对于YXHW证的准确率为72.5%,对于QYLX的准确率为96.7%。该模型对于肺结核三证候的分型具有较好的鉴别能力,盲法验证对于FYX证辨证的准确率为76.2%,对于YXHW证的准确率为70.8%,对于QYLX的准确率为80.0%。对筛选到的蛋白差异峰进行了鉴定和抗体芯片的验证,结果表明9416.7m/z蛋白峰是apolipoprotein C-Ⅲ载脂蛋白C-Ⅲ中的一段多肽。
结论：(1)筛选归纳了肺结核各证候特有的差异蛋白峰,可能是证候外在表现的内在病理机制和物质基础,为整体性评价肺结核中医证候的实质提供证据；归纳了7个蛋白峰在三证间的含量呈肺阴虚→阴虚火旺→气阴两虚逐渐降低变化,结果与中医理论关于肺结核证候病情演变相契合,差异的标志蛋白峰体现了肺结核证候间一定的相互关系及演变规律。(2)构建了肺结核中医证候蛋白辨证模型,为肺结核证候诊断的客观化提供蛋白质标准,有助于规范肺结核中医治疗的辨证施治。(3)鉴定和验证了载脂蛋白C-Ⅲ在肺结核三证候中差异表达,可能为肺结核中医证候分型的血清潜在标志物之一,可在临床和功能方面作进一步的验证。
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.
引文
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