^(18)O稳定同位素标记定量蛋白质组研究技术的建立与优化  

Establishment and Optimization of Stable Isotope ^(18)O-labeling Strategy for Quantitative Proteomics Research

作　　者：钱林艺[1] 应万涛[1] 刘新[1] 卢庄[1] 蔡耘[1] 何建勇[2] 钱小红[1] 

机构地区：[1]北京放射与辐射医学研究所 [2]沈阳药科大学,沈阳110015

出　　处：《分析化学》2007年第2期161-165,共5页Chinese Journal of Analytical Chemistry

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

摘　　要：建立了18O稳定同位素标记方法,用于复杂体系蛋白质相对定量分析。对影响蛋白质标记稳定性的实验条件进行了比较和优化。结果表明,采用酶切后标记的方法,酶切肽段在胰酶催化下,在pH 5.0的K2HPO4/KH2PO4缓冲体系中,37℃18O标记反应16 h,绝大部分肽段即可达到100%的标记效率。对多个16O/18O成对肽段峰强度的动态范围及定量准确度进行了考察。结果表明,18O标记方法是一种简便、稳定、可靠的相对定量方法,10倍动态范围内,标记率相对标准偏差在18.4%以内,16O/18O峰强度呈很好的线性关系。本实验考察了标记后的肽段在不同溶液体系中的稳定性,为复杂样品的预处理和预分离的溶液条件提供了依据。A method for relative quantitation of complex protein mixtures was developed with oxygen-18 labeling and mass spectrometric analysis. Some major influencing factors, such as pH, temperature and reaction time, were evaluated and optimized. The experimental results showed that a desirable labeling could be real- ized with tryptic catalyzing in a pH 5.0 buffer composed of K2 HPO4/KH2P04 for 16 hours at 37℃. Under these conditions, the efficiency of ^18O-labeling for the tryptic peptides could be up to 100%. The investigation on the dynamic range and quantitative accuracy were performed by observing and calculating the ratio of the peak intensity in mass spectra with several ^16O/^18O peptide couples from tryptic digestion of myoglobin and a favorable linear correlation with 0.99 of R2 value and 18.4% of deviation in a 1 ： 10 concentration range were obtained, which indicates that the protocol developed by the study was reliable and stable.

关 键 词：稳定同位素标记 ^18O 定量蛋白质组学 质谱 液相色谱 

分 类 号：Q51-33[生物学—生物化学]