Citation: LI Nannan, ZHOU Lianqi, MAO Xinli, ZHANG Jiao, WEI Junying, LIN Hongjun, LI Jiabin, TIAN Fang, ZHANG Yangjun, QIAN Xiaohong. A novel method for absolute protein quantification using ¹⁸O isotope labeled concatamers of Q peptides combined with isotope dilution-multiple reaction monitoring mass spectrometry[J]. Chinese Journal of Chromatography, ;2013, 31(6): 522-530. doi: 10.3724/SP.J.1123.2013.03057 shu

A novel method for absolute protein quantification using ¹⁸O isotope labeled concatamers of Q peptides combined with isotope dilution-multiple reaction monitoring mass spectrometry

LI Nannan ^1,2 ,
ZHOU Lianqi ² ,
MAO Xinli ² ,
ZHANG Jiao ² ,
WEI Junying ² ,
LIN Hongjun ² ,
LI Jiabin ² ,
TIAN Fang ² ,
ZHANG Yangjun ^2,, ,
QIAN Xiaohong ²

1.
1. Guangxi Medical University, Nanning 530021, China;

Corresponding author: ZHANG Yangjun,
Received Date: 31 March 2013
Available Online: 3 May 2013
Fund Project: 国家重大科学计划项目(2012CB910603,2010CB912704) (2012CB910603,2010CB912704)国家重大科学仪器设备开发专项项目(2011YQ030139,2011YQ06008408,2011YQ09000504,2012YQ12004407) (2011YQ030139,2011YQ06008408,2011YQ09000504,2012YQ12004407)国家高技术研究发展计划(2012AA020202) (2012AA020202)国家自然科学基金重点项目(20735005,21275159,31100591). (20735005,21275159,31100591)

A method of concatamers of Q peptides (QconCATs) protein labeled with ¹⁸O-multiple reaction monitoring mass spectrometry for absolute quantification of proteins is established. The purity of the QconCAT recombinant protein was characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and its purity was above 99%. The relative molecular mass was approximately 63.4 kDa. The peptides digested from the QconCAT recombinant protein and the extract of Thermoanaerobacter tengcongensis (TTE) were analyzed by mass spectrometry. The raw data were processed by pFind and pLabel softwares. The results showed that the efficiencies of protein digestion and the ¹⁸O labeling efficiency were able to meet the need of the protein quantification. The performance of the method was evaluated. The absolute contents of the selected proteins in TTE were determined with the relative standard deviations of less than 20% and the accuracy is high. The method not only avoid using the expensive reagent of stable isotope labeling with amino acids in cell culture (SILAC), but also provides an alternative way for the accurately absolute quantification of proteins in biological samples for quantitative proteomic research.
- concatamers of Q peptides (QconCATs),
- isotope dilution,
- multiple reaction monitoring mass spectrometry,
- ¹⁸O labeling,
- proteomics
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A novel method for absolute protein quantification using 18O isotope labeled concatamers of Q peptides combined with isotope dilution-multiple reaction monitoring mass spectrometry

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通讯作者: 陈斌, bchen63@163.com

A novel method for absolute protein quantification using ¹⁸O isotope labeled concatamers of Q peptides combined with isotope dilution-multiple reaction monitoring mass spectrometry