Non-depleted human plasma proteome of pulmonary tuberculosis

Novel biomarkers to identify infectious patients transmitting Mycobacterium tuberculosis are urgently needed to control the global tuberculosis (TB) pandemic. We hypothesized that proteins released into the plasma in active pulmonary TB are clinically useful biomarkers to distinguish TB cases from healthy individuals and patients with other respiratory infections. We applied a highly sensitive non-depletion tandem mass spectrometry discovery approach to investigate plasma protein expression in pulmonary TB cases compared to healthy controls in South African and Peruvian cohorts. . Plasma samples were analysed from 11 untreated male patients with active pulmonary TB and 10 male healthy control samples. Each plasma sample was initially separated into four segments by size exclusion chromatography, and each segment was processed individually. Analyses of plasma segments were performed in twelve iTRAQ (isobaric tags for relative and absolute quantification) 8-plex experimental sets in a block randomised design comprising three experimental sets. Each iTRAQ experiment contained a bridging master-pool plasma sample run in every experiment. Healthy controls were matched to TB samples by age, ethnicity, and smoking status within each iTRAQ set. Protein abundances from the plasma segments and multi-consensus reports were combined and adjusted for experimental batch effects. Protein abundances from the remaining combined plasma segment proteomes between experimental sets and the combined multi-consensus proteomes were analysed by complementary bioinformatic approaches to identify candidate diagnostic protein biomarkers. In total, 4,696 protein identifications were made across all iTRAQ experiments, at 5% FDR (false discovery rate). This comprised 2,332 unique host-derived proteins and 22 Mtb-derived proteins. Of these, 594 host proteins had a quantification result for every sample analysed and therefore comprised the complete quantified proteome. Subsequent bioinformatic analysis identified 118 differentially expressed proteins by three complementary bioinformatic pipelines and were taken forward in subsequent validation work as strong candidate biomarkers of pulmonary TB.

为控制全球结核病（TB）大流行，亟需开发可识别结核分枝杆菌（Mycobacterium tuberculosis）传播感染者的新型生物标志物。本研究假设，活动性肺结核患者释放入血浆的蛋白质可作为区分结核病患者、健康个体与其他呼吸道感染患者的临床实用生物标志物。本研究针对南非与秘鲁队列中的肺结核患者与健康对照，采用高灵敏度非耗尽型串联质谱发现策略，探究血浆蛋白质表达谱差异。本研究分析了11例未经治疗的活动性肺结核男性患者与10例男性健康对照的血浆样本。每份血浆样本首先通过尺寸排阻色谱分为四个组分，随后对每个组分分别进行处理。血浆组分的分析在12组iTRAQ（同量异位素相对与绝对定量标签，isobaric tags for relative and absolute quantification）8重标记实验集中开展，实验采用区组随机化设计，每个区组包含3个实验集。每一组iTRAQ实验均设置桥接混合血浆样本，且该样本在每一次实验中均参与检测。在每组iTRAQ实验中，健康对照样本均按照年龄、种族与吸烟状态与肺结核样本进行匹配。将血浆组分的蛋白质丰度数据与多共识报告进行整合，并针对实验批次效应进行校正。针对实验集间剩余的整合血浆组分蛋白质组数据，以及整合后的多共识蛋白质组数据，本研究采用互补生物信息学方法开展分析，以筛选潜在的诊断用蛋白质生物标志物。所有iTRAQ实验中共鉴定到4696个蛋白质，错误发现率（FDR，false discovery rate）控制在5%。其中包含2332个独特的宿主源性蛋白质与22个结核分枝杆菌源性蛋白质。在此之中，有594个宿主蛋白质在所有分析样本中均获得定量结果，因此构成了完整的定量蛋白质组。后续通过三种互补生物信息学分析流程开展生物信息学分析，最终筛选得到118个差异表达蛋白质，这些蛋白质作为肺结核的潜在强效生物标志物进入后续验证工作。