[A novel method for absolute protein quantification using 18O isotope labeled concatamers of Q peptides combined with isotope dilution-multiple reaction monitoring mass spectrometry].
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.
Guangxi Medical University, Nanning 530021, China.
Nannan
N
Li
Lianqi
L
Zhou
Xinli
X
Mao
Jiao
J
Zhang
Junying
J
Wei
Hongjun
H
Lin
Jiabin
J
Li
Fang
F
Tian
Yangjun
Y
Zhang
Xiaohong
X
Qian
D004591:Electrophoresis, Polyacrylamide Gel; D007553:Isotope Labeling; D013058:Mass Spectrometry; D010455:Peptides; D011506:Proteins / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins; D046968:Thermoanaerobacter
D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; C090695:peptide Q