Evaluation of the accuracy of protein quantification using isotope TMPP-labeled peptides.
N-succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl) phosphonium bromide (TMPP-Ac-OSu) reacts rapidly, mildly, and specifically with the N-terminals of proteins and peptides. Thus, it can be developed as an ideal isotope-coded tag to be used in quantitative proteomics. Here, we present a strategy for light and heavy TMPP-based quantitative proteomic analysis, in which peptides in a mixture can be quantified using an on-tip TMPP derivatization approach. To demonstrate the accuracy of this strategy, light and heavy TMPP-labeled peptides were combined at different ratios and subsequently analyzed by LC-MS/MS. The MS spectra and scatter plots show that peptide and protein ratios were both consistent with the mixed ratios. We observed a linear correlation between protein ratios and the predicted ratios. In comparison with SILAC method, the TMPP labeling method produced similarly accurate quantitative results with low CVs. In conclusion, our results suggest that this isotope-coded TMPP method achieved accurate quantification and compatibility with IEF-based separation. With the inherent advantages of TMPP derivatization, we believe that it holds great promise for future applications in quantitative proteomics analysis.
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, P. R. China.
Hongyan
H
Shen
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, P. R. China.
Mingrui
M
An
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, P. R. China.
Xiao
X
Zou
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, P. R. China.
Xuyang
X
Zhao
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, P. R. China.
Qingsong
Q
Wang
Institute of Organic Chemistry, College of Chemistry, Beijing Normal University, Beijing, P. R. China.
Guowen
G
Xing
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, P. R. China.
Jianguo
J
Ji
D000818:Animals; D002347:Carps / Q000378:metabolism; D002364:Caseins / Q000032:analysis; D002853:Chromatography, Liquid; D029941:Fish Proteins / Q000032:analysis / Q000378:metabolism; D005910:Glioma / Q000378:metabolism; D045325:HCT116 Cells; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D051379:Mice; D009861:Onium Compounds / Q000737:chemistry*; D009943:Organophosphorus Compounds / Q000737:chemistry*; D010455:Peptides / Q000032:analysis* / Q000737:chemistry*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D051381:Rats; D053719:Tandem Mass Spectrometry / Q000379:methods; D014407:Tumor Cells, Cultured
D002364:Caseins; D029941:Fish Proteins; C000608117:N-succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium; D009861:Onium Compounds; D009943:Organophosphorus Compounds; D010455:Peptides
Isotope-coded peptide labeling; On-tip derivatization; StageTip; TMPP-Ac-OSu; Technology