血液样本蛋白质组分析方法的比较研究

Comparative Study on Methods of Analyzing Proteome in Blood Samples

比较和优化血液样本蛋白质组学样品制备和质谱分析技术,为深度研究和挖掘血液样本蛋白质组学信息创造条件。采用Q-Exactive Plus质谱仪,对比分析血浆、血清和去除高丰度蛋白血清预处理方法制备的大鼠血样蛋白质组构成;比较血清样本的常规酶切、45℃孵育、热辅助酶切、二次热辅助酶切、尿素辅助酶切和变温酶切的效率;比较数据依赖性采集(data-dependent acquisition,DDA)、数据非依赖性采集(data independent acquisition,DIA)和平行反应监测(parallel reaction monitoring,PRM)质谱数据采集的定性定量特征;采用优化的方法进行大鼠血液样本蛋白质组分析。血清样本去除高丰度蛋白后蛋白鉴定数更高、定量重复性更好;热辅助酶切和变温酶切血清样品的蛋白和肽段鉴定数以及质谱谱图匹配率相对较高,蛋白酶切效率和定性定量重复性较好;DDA操作简便,DIA重复性高,PRM定量精确;血清样本去除高丰度蛋白,采用热辅助结合变温酶切和DDA数据采集模式,3次重复试验分别鉴定到490、490、504个蛋白,鉴定总蛋白数590个,共有蛋白占比69.8%。优化的方法操作简单,蛋白鉴定率较高,重复性好,适用于血液样本的蛋白质组学分析。

The objective is to compare and optimize the technologies of blood sample preparation and mass spectrometry analysis,for further studying and mining proteomic information in blood samples.A Q-Exactive Plus mass-spectrometer was used to compare the proteome composition of R.norvegicus blood samples prepared from plasma,serum and serum with high-abundance protein removed.The efficiency of trypsin digestion of serum proteins was also investigated by using conventional digestion,45℃digestion,heat assisted digestion,repeated heat assisted digestion,urea assisted digestion and variable temperature digestion.Then the qualitative and quantitative characteristics of three mass spectrometry analysis methods,Data-Dependent Acquisition(DDA),Data Independent Acquisition(DIA)and Parallel Reaction Monitoring(PRM)were compared.Finally,the blood sample of R.norvegicus was analyzed based on optimized proteomic technologies.After removing high-abundance protein from serum samples,the number of identified proteins was higher and the quantitative repeatability was better.The number of identified proteins and peptides,and the rate of matched mass spectrum were relatively high when serum samples digested by heat assisted digestion and variable temperature digestion.The efficiency of these two digestion methods and the repeatability of their qualitative and quantitative data were acceptable.Compared the three mass spectrometry methods,DDA was more convenient,DIA was highly reproducible and PRM was more accurate.The 490,490 and 504 proteins were identified in 3 repeated experiments respectively when high-abundance protein was removed from serum samples and digested proteins with heat assisted-variable temperature digestion and collected data by DDA method.While a total of 590 proteins were detected,and repetition rate of identified protein was 69.8%.In conclusion,the optimized method has advantages of simple operation,high protein identification rate and good repeatability,and is suitable for proteomic analysis of blood samples.