福尔马林固定石蜡包埋组织样本的蛋白质组制备技术研究  

Proteomic Preparation Techniques for Formalin-Fixed Paraffin-Embedded Tissue Samples

作　　者：陆澳 孟波 赵佳威 廖焕玥 叶子弘 方向[2] 赵洋 LU Ao;MENG Bo;ZHAO Jia-Wei;LIAO Huan-Yue;YE Zi-Hong;FANG Xiang;ZHAO Yang(College of Life Sciences,China Jiliang University,Hangzhou 310018,China;National Market Supervision Technology Innovation Center(Mass Spectrometry),Center for Advanced Measurement Science,National Institute of Metrology,Beijing 100029,China)

机构地区：[1]中国计量大学生命科学学院,杭州310018 [2]中国计量科学研究院前沿计量科学中心,国家市场监管技术创新中心(质谱),北京100029

出　　处：《分析化学》2025年第1期84-93,共10页Chinese Journal of Analytical Chemistry

基　　金：科技基础资源调查专项项目(No.2022FY101202);国家重点研发计划项目(No.2022YFF0608404);国家自然科学基金项目(No.21927812);中国计量科学研究院基本业务费重点项目(No.AKYZD2111)资助。

摘　　要：基于不同的脱蜡液(Triton X-100和二甲苯)、裂解液(TFE和RapiGest)以及酶切方案(iST、SP3和FASP),通过正交实验设计了12种福尔马林固定石蜡包埋(FFPE)组织样本的蛋白质组前处理技术方案,探索了FFPE蛋白质组样本制备的最佳实验条件。从蛋白质和肽段的鉴定深度、鉴定稳定性和蛋白丰度的定量水平等方面进行了系统性评估,研究结果显示,Triton X-100脱蜡液和二甲苯对蛋白质组的鉴定影响较小,而TFE裂解液和iST酶切方案均能显著提升FFPE组织样本的蛋白质组分析水平。鉴于二甲苯具有的潜在毒性,最终确定以Triton X-100脱蜡液、TFE裂解液和iST酶切方案为基础的TTI技术方案为最优选择。此方案显示了最佳的重复性和稳定性,同时能够鉴定更多与重要生物学功能相关的特异性蛋白质。本研究建立的TTI技术方案为FFPE组织样本的蛋白质组分析提供了一种高效且全面的方法,显著提升了样本的蛋白质组鉴定的重复性和稳定性。Twelve pre-processing protocols for formalin-fixed paraffin-embedded(FFPE)tissue samples were developed by orthogonal experimental design,incorporating different dewaxing buffers(Triton X-100 and xylene),lysis buffers(TFE and RapiGest),and enzyme digestion methods(iST,SP3,and FASP)to explore the optimal experimental conditions.These protocols were assessed based on protein and peptide identification depth,identification stability,and quantitative levels of protein abundance.The results indicated that Triton X-100 and xylene minimally impacted proteomics identification,whereas the TFE lysis buffer and iST digestion method significantly enhanced the proteomics analysis of FFPE samples.Considering the potential toxicity of xylene,the TTI protocol based on Triton X-100,TFE,and iST was determined to be the optimal choice.This protocol exhibited the best repeatability and stability,and a higher number of proteins associated with significant biological functions were identified.In conclusion,the established TTI protocol offered an efficient and comprehensive approach for proteomic analysis of FFPE samples,significantly enhancing the repeatability and stability of protein identification.

关 键 词：福尔马林固定石蜡包埋样本 样本制备 蛋白质组 质谱 液相色谱-串联质谱联用 

分 类 号：TQ936[化学工程] O657.63[理学—分析化学]