微量蛋白质样品高效提取及分离方法研究

Protein is directly related with various physiological and pathological states of organisms. It is known that most of the reported drug targets and disease-specific biomarkers are proteins, and now proteomics is the central of biology studies. Liquid chromatography coupled with mass spectrometry (LC-MS) is the core equipment of current proteomics. In the previous works, we have developed various technologies to improve the separation resolution, detection sensitivity, analysis throughput, and proteome coverage of the LC-MS system for tryptic peptides analyses. And monolithic columns and isotope labeling technologies were successfully applied in tryptic peptides' identification and quantification. An online system integrated with isotope labelling, multidimensional separation, and MS detection was developed and applied for disease biomarkers' discovery. As there is lack of high efficient protein extraction and separation technologies and methods, we propose to optimize the composition of protein extraction buffer to increase the extraction efficiency for limited amount of protein samples, develop capillary liquid chromatography (LC) systems for intact protein separation with high resolution by using different types of monolithic columns and construct an automated Top-Down proteome analysis system integrated with sample preparation, protein LC separation and tandem mass spectrometry (MS/MS) detection. The outcome of this project will increase the throughput and sensitivity greatly in Top-Down proteomics, which is extremely important for comprehensive proteome analysis.

蛋白质是生物体各种生物学功能的具体执行者，蛋白质组学研究已经成为后基因组时代推动生命科学发展的关键之一。液相色谱-质谱联用是现代蛋白质组学研究的核心仪器装备。申请人在前期研究工作中致力于提高现有液质联用系统对酶解多肽样品的分离分辨率、检测灵敏度、分析通量和蛋白质组覆盖率，构建了在线同位素标记-多维色谱分离-质谱检测全自动化蛋白质组定量分析集成化系统。针对现阶段微量蛋白质样品高效提取分离技术的缺乏，本项目拟发展针对大分子蛋白质的高效蛋白质样品提取方法，高分辨毛细管液相色谱分离系统并集成样品预处理功能，显著提高现有Top-Down 蛋白质组定性定量分析技术的灵敏度、通量和覆盖率。本项目的开展实施将有效推动我国在微量蛋白质样品高效提取和分离方面的研究进展。

本项目主要针对目前蛋白质组分析技术中蛋白质样品高效提取分离方面的不足而开展，总体目标为提高微量组织和细胞样品中蛋白质提取效率，发展高效的蛋白质分离色谱体系，并且完成与质谱检测的集成化。通过三年的的努力，项目组完成了项目任务，具体如下：1）在微量组织和细胞样品提取方面，发现1%十二烷基麦芽糖苷（DDM）结合8 M尿素缓冲溶液可以从组织和细胞样品中有效提取糖基化蛋白质（多为疏水性膜蛋白质）。由于DDM与胰蛋白酶酶切、色谱分离、质谱检测等高度兼容，提取蛋白质样品无需纯化可直接进行分析检测，从鼠脑组织中鉴定到的糖基化位点数目是常规方法的两倍以上；成功合成了糖肽树状聚合物修饰高亲水性磁球，其亲水相互作用富集糖基化蛋白质和多肽的能力较常规亲水材料提高了5倍，对复杂样品糖基化位点鉴定数目提高了56%；2）在整体蛋白质高效色谱分离和质谱检测方面，采用三氟乙酸液相色谱分离体系，并利用密闭型质谱离子源调节酸性气氛消除三氟乙酸的离子抑制效应，实现了色谱分离分辨率提高36%，质谱检测信号提高10倍以上，在大肠杆菌整体蛋白质提取样品分析中将有效质谱检测信号数目提高了96%；3）在蛋白质高灵敏度度分析系统和方法集成方面，采用多酶酶切策略进行人肝提取蛋白质样品酶切，亲水作用色谱法和酰肼化学法高效富集糖肽，在线多维色谱对富集糖肽进行高分辨色谱分离，并使用两种不同类型质谱进行糖肽分析鉴定，建立了一种复杂组织样品蛋白质糖基化大规模鉴定策略，从人肝组织样品中鉴定到4793个糖基化位点，这是迄今为止最大的人源蛋白质糖基化数据集；发展了一种基于反相-强阳离子交换-反相三维液相色谱分离的样品处理方法，样品纯化、二甲基标记和预分级都在同一根色谱柱上进行，显著消除了样品的损失和污染，共得到了2759个蛋白的定量结果。目前项目已经发表了通讯作者SCI论文11篇，其中影响因子5以上论文5篇，包括Analytical Chemistry 2篇，Chemical Communications 3篇，Journal of Proteome Research 2篇，Journal of Chromatography A 2篇，Analytica Chimica Acta 1篇，Analytical and Bioanalytical Chemistry 1篇，申请中国发明专利3项，在国内外学术会议上做特邀/口头报告14次。