基于MRM靶向定量的蛋白分析技术在疾病生物标志物精准检测中的应用——以chemerin为例

Proteins participate in almost of all life cycles, and the changes in expression levels of functional protein have close relationship to kinds of physiological and pathological circumstances. The analytical approaches in pharmaceutical analysis are influential to the discovery of new biomarkers. MRM-based targeted proteomic analytical method is well accepted and widely used in quantitation of proteins. Chemerin is recognized as an inflammation related adipokine which plays an important role in immune-mediated inflammatory disease and metabolic disease. It exists in several isoforms with different biological activity, but the current biological methods of detection could not ensure the accuracy, nor to identify the specific protein isoforms. In the preliminary trail, signals of several isoform-specified unique peptides, including new isoforms, have been clearly identified by LC/MRM-MS in human follicular fluid sample. Here this program will be carried out based on optimizing the protein pretreatment technology including enzymatic digestion and enrichment methods, followed by establishment of the targeted quantitative LC-MS protein analysis strategy. And the result will be validated by biological strategies. The method will be employed to analyze different human bio-fluids such as serum, follicular fluid and synovial fluid from patient suffering different diseases including but not limited to rheumatoid arthritis and polycystic ovary syndrome, as well as the healthy control. This study will reveal the specific chemerin isoform related to different diseases. These could be defined as real biomarker which assists clinical diagnosis and treatment.

蛋白质直接参与了所有生命周期，功能性蛋白表达量的变化与各种生理病理状态紧密相关。药物分析学提供的分析检测技术对各类生物标志物的发现有重要影响，基于三重四极杆质谱MRM模式的靶向定量蛋白质分析技术，是当前目标蛋白定量分析的权威策略。Chemerin作为一类与炎症相关的脂肪因子，在自身免疫疾病和代谢疾病中起重要作用，其不同亚型生理活性差异巨大，当前主流的生物检测手段既不能保证分析结果的准确性，也无法区分具体的蛋白亚型。预实验时我们利用LC/MRM-MS在人卵泡液样本中检测到多种亚型的专属肽段信号，包括未报道过的全新亚型。本课题将在优化酶解与富集等前处理技术的基础上，建立chemerin蛋白靶向定量分析液质联用方法，运用生物检测手段辅助验证，以类风湿性关节炎和多囊卵巢综合征等疾病患者为研究对象，对血清、关节滑液、卵泡液等样本进行精准检测，明确作为疾病相关生物标志物的具体亚型，辅助临床诊断与治疗。

背景：Chemerin作为一类与炎症相关的脂肪因子，在自身免疫疾病和代谢疾病中起重要作用，其不同亚型生理活性差异巨大，当前主流的生物检测手段既不能保证分析结果的准确性，也无法区分具体的蛋白亚型。本项目在预试验时利用LC/MRM-MS在人卵泡液样本中检测到了多种亚型的专属肽段信号，包括未报道过的全新亚型。.主要研究内容：1.探索优化了样品前处理条件：包括酶种类、酶与底物比例、孵育时长、温度、pH值、终止反应条件等。2.建立并验证了基于MRM模式的LC-MS分析方法：对特异性肽段进行子离子扫描并结合软件计算预测结果，选择定量离子对与辅助定性离子对，优化分析时间与碰撞能，合成非放射性重同位素标记肽，作为内标加入不同样本根据质谱峰面积比进行绝对定量，开展方法学研究，包括标准曲线、回收率、精密度、稳定性、检测限、定量限等。3.探究免疫亲和沉淀富集不同样品中chemerin蛋白的方法：将chemerin单克隆抗体固定在交联珠状琼脂糖柱床，特异性吸附纯化血液、卵泡液、关节滑膜液、尿液等生物样本中的chemerin蛋白，采用western blot蛋白免疫印迹分析法验证富集所得蛋白的含量。4.检测不同疾病的不同体液样本：通过LC-MS分析血清、关节滑液、卵泡液（多囊卵巢综合征患者）等临床样本，发现与类风湿性关节炎、多囊卵巢综合征相关的具体chemerin亚型。5.在大鼠模型上探索筛选验证治疗多囊卵巢综合症的中药活性成分。.重要结果、关键数据及其科学意义：确定了体液样品前处理条件，建立并验证了基于LC/MRM-MS的chemerin蛋白靶向定量液质联用分析方法，检测疾病与对照组体液（包括108份卵泡液、136份血清、以及35份关节滑液），发现多囊卵巢综合征患者156F和157S两个亚型水平明显增高，而155A、156F和157S三种亚型与类风湿性关节炎密切相关，可以作为疾病诊治相关的生物标志物。