利用定量蛋白组学对脓毒症外周血单个核细胞差异蛋白的研究

Quantitative proteomic reveals the differences of peripheral blood mononuclear cells in the host immune response to sepsis

目的 探讨基于数据非依赖性采集高分辨率色谱- 质谱技术（data independent acquisition liquid chromatography-mass spectrometry, DIA LC-MS）对于脓毒症外周血差异蛋白研究的可行性。方法 采用前瞻性研究，纳入2016 年4 月至2016 年7 月复旦大学附属上海市第五人民医院重症监护室（ICU）收治的10 例脓毒症以及10 例同时期入住ICU 年龄性别相仿的术后未并发脓毒症的患者（疾病对照组）。采用最新的数据非依赖性采集技术（data independent acquisition，DIA）联合skyline 数据提取软件对10 例脓毒症和10 例对照组的外周血单个核细胞的蛋白进行研究分析，所得的数据分别采用主成分分析法（principal component analysis，PCA）、偏最小二乘法- 判别分析（partial least squares discriminant analysis，PLS-DA）和正交偏最小二乘法判别分析（orthogonal partial least squares discriminant analysis，OPLS-DA）进行模式识别分析，确定外周血单个核细胞的差异蛋白，进行通路分析以及GO 分析。根据PLS-DA 模型变量的重要性投影（variable importance in the projection, VIP 值）初步筛选可能的蛋白标志物，并对贡献最大（VIP 〉1, P〈0.05）的3 个蛋白进行ELISA 验证和ROC 曲线的分析。结果 总共鉴定到了1 062 个碎片离子加和得到119 个差异蛋白，其中31 个蛋白上调，88 个蛋白下调。通过通路分析发现与碳代谢，血小板激活，细菌侵袭上皮，补体凝血瀑布激活有关。其中高迁移率蛋白1（HMGB-1），中性粒细胞明胶酶相关脂质运载蛋白（NGAL），基质金属蛋白酶8（MMP-8）用ELISA 验证在脓毒症与疾病对照组的外周血单个核细胞表达中差异有统计学意义（P〈0.01）。ROC 曲线下面积均大于0.85，有较好的敏感度和特异度。结论 利用DIA-MS 技术对脓毒症外周血单个核细胞进行研究检测，PLS-DA、OPLS-DA、PCA 模式识别均能用于蛋白组学数据的分析，初步筛选HMGB-1、NGAL、MMP-8 是值得进一步研究的脓毒症外周血单个核细胞候选生物标志物。

Objective To investigate the feasibility of differential expression proteins identification in peripheral blood mononuclear cells （PBMCs） of sepsis patients using Data Independent Acquisition liquid chromatography-mass spectrometry （DIA LC-MS）. Methods Prospective studies were employed and targeted at 10 sepsis patients admitted to the Intensive Care Unit （ICU） of Shanghai Fifth People＇s Hospital Affiliated to Fudan University from April 2016 to July 2016. And 10 patients admitted to the ICU with similar age and sex that were not complicated with sepsis were served as the control group.The proteins from peripheral blood mononuclear cells in 10 sepsis patients and 10 control persons were analyzed using the latest DIA LC-MS technology and the skyline data extraction software; the model of data obtained from the above analysis was further discriminatorily analyzed with principal component analysis （PCA）, partial least squares discriminant analysis （PLS-DA） and orthogonal partial least squares discriminant analysis （OPLS-DA）; then the differential proteins of peripheral blood mononuclear cells were analyzed by Pathway analysis and GO analysis. The possible markers were identified by preliminary screening according to variable importance in the projection （VIP）. The top 3 proteins （VIP 〉 1, P〈 0.05） were verified by ELISA and their ROC curves were analyzed. Results Totally 1 062 fragment ions were identified and 119 proteins were obtained. Among them, 31 proteins were up-regulated and 88 proteins down-regulated. Pathway analysis showed that carbon metabolism, platelet activation, bacterial invasion of epithelium, and complement coagulation cascade activation were participated in the development of sepsis. ELISA showed that significant difference of HMGB-1, MMP-8, and LCN2 in the expression of peripheral blood mononuclear cells in the sepsis and control people （P〈0.01）. The area under the ROC curve is greater than 0.85, which has good sensitivity and specificity. Conclusions DIA-MS is a compelling way for detecting differential expression proteins. PCA, PLSDA and OPLSDA are suitable for pattern recognition. The high expression of HMGB-1, NGAL and MMP-8 in immune cells may be the potential biomarker of the disease, which lays the foundation for research of early diagnosis and treatment of sepsis.