蛋白组学定量新方法.pptx

生物质谱研讨会 复旦大学 2014-07-03蛋白质组定量分析新技术新方法Novel Methods and Techniques for Proteome Quantification张玉奎 张丽华中国科学院大连化学物理研究所Dalian Institute of Chemical Physics, Chinese Academy of Sciences 报告内容 Outline背景 Background准等重二甲基化标记定量新方法 Pseudo-isobaric dimethyl labeling method 集成化蛋白质组定量分析平台 Integrated platform for proteome quantification背景 Background基于二级质谱的定量方法MS/MS based proteome quantification基于报告离子 reporter ions based method 标记效率高，准确度低 high labeling efficiency, poor accuracy 基于成对碎片离子 pair fragment ions based method 准确度高，标记效率低 high accuracy, low labeling efficiency解决办法 Our strategy 准等重二甲基化标记定量新方法 Pseudo-isobaric dimethyl labeling method 1.准等重二甲基化标记蛋白质组定量方法Pseudo-isobaric Dimethyl labeling method (pIDL)甲醛标记引入标记试剂的种类多，标记效率高，但是目前二甲基化定量方法存在定量的动态范围小、定量精密度差等不足Dimethyl labeling is widely used for proteome quantification due to high labeling efficiency and various reagents, but it suffers from low dynamic range and precision.由于核结合能的不同导致的质量亏损，通过高分辨率的质谱可以在MS2分辨这微小差异，进而可以在宽线性范围内实现蛋白质组的准确定量The subtle mass differences, arising from the fact that nuclear binding energy, can be resolved by high MS/MS resolution. High accurate proteome quantification in wide dynamic range can be realized by the paired fragment ions AtomAtomic WeightMass Defect/mDaH1.007 825+6.17D2.014 10112C12+3.3513C1314.003 074-3.0015N15.000 108Zhang et al. Anal. Chem., 2013, 85, 10658－10663实验流程 Workflow选择Lys-C蛋白酶酶解蛋白，分别用 13CD2O 和NaCNBH3用作轻标记， CD2O 和NaCNBD3 用作重标记Lys-C protein digests were labeled with 13CD2O and NaCNBH3, in addition to CD2O and NaCNBD3, as light and heavy isotopologues, respectively.一级谱相差11.68 mDa，在常用分辨率下无法实现分辨The mass differences were 11.68 mDa, which is indistinguishable at the MS level. 碎片离子存在5.84 mDa质量差异，当MS/MS选择高分辨率时，碎片离子会成对出现，从而用于蛋白质定量分析High-resolution MS/MS is capable of resolving fragment ions with a mass difference of 5.84 mDa in low mass range (m/z400), which allows proteome quantification.重标轻标One signal准等重二甲基化标记蛋白质定量方法示意图Schematic representation pseudo-isobaric dimethyl labe