基于新型样品预处理和质谱全离子监测的蛋白组深度覆盖定量方法研究

To solve the problem of the low coverage of quantitative proteomics which is caused by the severe loss of low abundance proteins during the sample preparation and the suppression effect on low abundance proteins during LC-MS/MS detection, we will intend to perform the following two aspects research work respecting to sample preparation and mass spectrometry analysis: 1) To improve the solubility and digestion performance of hydrophobic proteins analysis and reduce the severe loss of low abundance proteins during the off-line pretreatment process, we will develop ionic liquid based in-situ filter-aided sample pretreatment method; 2) To improve the sensitivity and accuracy of low-abundance proteins quantification, we will combine liquid chromatography fractionation and mass spectrometry gas phase fractionation to develop a novel quantification method, which targets to monitor the whole MS/MS spectra, in order to reduce the suppression effect of high abundance proteins and increase the MS/MS quantitative information of low-abundance proteins. By using the newly developed method, we will comprehensively analyze the differentially expressed proteins from human liver cancer cell lines with high and low metastasis ability, to provide important technique support for screening the target proteins associated with tumor metastasis and elucidate the mechanism for tumor metastasis.

针对由于低丰度蛋白质在样品预处理过程中损失严重，以及在液质联用中难以被检测而导致的蛋白质组定量分析覆盖度低的问题，本项目拟以样品预处理和质谱定量信息采集为切入点：1）发展基于离子液体的原位蛋白质组样品预处理新方法，解决膜蛋白质等低丰度蛋白质提取困难、酶解效率差以及在多步离线处理过程中损失严重等问题；2）将液相色谱分级和质谱气相分级相结合，发展基于全离子监测的二级谱定量方法，提高低丰度蛋白质定量的灵敏度和准确度。将发展的新方法用于实现人肝癌高、低转移细胞株的蛋白质组深度覆盖定量分析，为筛选与肝癌转移相关的蛋白质，并阐明肿瘤的转移机理提供重要技术支撑。

针对由于低丰度蛋白质在样品预处理过程中损失严重，以及在液质联用中难以被检测而导致的蛋白质组定量分析覆盖度低的问题，本项目以样品预处理和质谱定量信息采集为切入点， 1）建立了基于离子液体的原位蛋白质组样品预处理新方法，解决膜蛋白质等低丰度蛋白质提取困难、酶解效率差以及在多步离线处理过程中损失严重等问题，使得蛋白质的鉴定能力较基于SDS的FASP方法提高16%，显著提高了蛋白质的鉴定覆盖度； 2）将液相色谱分级和质谱气相分级相结合，发展了基于全离子监测的二级谱定量方法，可实现10000以上蛋白质的定量分析，将定量分析的丰度检测范围从常规的5-6个数量级提高到8个数量级，极大程度上提高了低丰度蛋白质定量的灵敏度和准确度。将发展的新方法用于实现人肝癌高、低转移细胞株的蛋白质组深度覆盖定量分析，发现了64个差异蛋白质，其中高达53% 均为膜蛋白质，并将目前尚未明确报道与肝癌转移相关的差异蛋白质表达量进行Western blot验证，生物学验证结果与基于质谱的蛋白质定量结果高度一致。随后，通过STRING分析，选取了节点蛋白质CALB2进行后续生物学分析，通过细胞水平和老鼠活体实验证明了CALB2对于肝癌转移的促进作用，为阐明肿瘤的转移机理提供重要的数据支撑。项目执行期间，共发表SCI论文6篇，会议论文5篇，申请发明专利7件，培养博士研究生3名。