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.
出处
《分析化学》
SCIE
EI
CAS
CSCD
北大核心
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
关键词
稳定同位素标记
^18O
定量蛋白质组学
质谱
液相色谱
Stable isotope labeling, oxygen-18, quantitative proteomics, mass spectrometry, liquid chromatography
作者简介
E-mail： qianxh@nic.bmi.ac.cn
