18 O同位素标记定量肽段串联体蛋白质结合同位素稀释-多反应监测质谱的蛋白质绝对定量新方法

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.