基于生物质谱的乙酸酐稳定同位素标记定量蛋白质组学方法的建立与优化  

Establishment and Optimization of Acetic Anhydride Stable Isotope Labeling Method Based on Mass Spectrometry in Quantitative Proteomics

作　　者：刘新[1] 应万涛[1] 周春喜[1] 蔡耘[1] 钱小红[1] 

机构地区：[1]北京放射与辐射医学研究所

出　　处：《分析化学》2007年第12期1687-1695,共9页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(No.20405017;No.20505018;No.20505019);北京市科技计划重大项目(No.H030230280190);国家重点基础研究发展规划项目(No.2004CB518707);国家自然科学基金重点项目(No.20635010);国家科技计划课题(No.2006CB0D1600)资助课题

摘　　要：建立了一种基于生物质谱的乙酸酐稳定同位素标记,定量蛋白质组学研究方法,优化了影响标记效率的各种条件。在pH8.0的Na2B4O7/H3BO3缓冲体系中,当乙酸酐摩尔浓度25倍过量于肽段摩尔量,22℃反应30 min时,标记即可完全。对多对H6/D6-乙酸酐标记肽段在基质辅助激光解吸电离质谱中的动态范围及定量准确度进行了考察,并通过串联质谱分析确定了乙酰化位点。结果表明:在10倍和30倍动态范围内,线性关系良好(r=0.99,r=0.98),理论值和观测值的偏差分别为0.5%和20%。A strategy for quantitative proteome analysis was established through H6/D6-acetic anhydride stable isotopic labeling. Several major influencing factors, such as reactive buffer system, acetic anhydride concentration, pH value, temperature and reaction time, were examined. The results showed that a satisfied acetic anhydride labeling could be realized in a pH 8.0 reactive buffer composed of Na2B4OT/H3BO4 when the reaction was kept for 30 minutes at 22℃, with a 25-fold molar excess of acetic anhydride over free amino groupscontained peptides. Under the optimized conditions, the efficiency of acetic anhydride labeling for the tryptic peptides was up to 100%. The investigation on the dynamic range and quantitative accuracy were performed by calculating the ratio of the peak intensity in mass spectra with several H6/D6-acetylated peptide couples. A good linearity was obtained within the dynamic ranges of 1：10 and 1：30, with the R^2value of 0.99 and 0.98 and RSD of 0.5% and 20% , respectively.

关 键 词：乙酸酐稳定同位素标记 定量蛋白质组学 生物质谱 

分 类 号：Q51-3[生物学—生物化学]