A novel quantitative proteomics workflow by isobaric terminal labeling.
Quantification by series of b, y fragment ion pairs generated from isobaric-labeled peptides in MS2 spectra has recently been considered an accurate strategy in quantitative proteomics. Here we developed a novel MS2 quantification approach named quantitation by isobaric terminal labeling (QITL) by coupling (18)O labeling with dimethylation. Trypsin-digested peptides were labeled with two (16)O or (18)O atoms at their C-termini in H(2)(16)O or H(2)(18)O. After blocking all ε-amino groups of lysines through guanidination, the N-termini of the peptides were accordingly labeled with formaldehyde-d(2) or formaldehyde. These indistinguishable, isobaric-labeled peptides in MS1 spectra produce b, y fragment ion pairs in the whole mass range of MS2 spectra that can be used for quantification. In this study, the feasibility of QITL was first demonstrated using standard proteins. An accurate and reproducible quantification over a wide dynamic range was achieved. Then, complex rat liver samples were used to verify the applicability of QITL for large-scale quantitative analysis. Finally, QITL was applied to profile the quantitative proteome of hepatocellular carcinoma (HCC) and adjacent non-tumor liver tissues. Given its simplicity, low-cost, and accuracy, QITL can be widely applied in biological samples (cell lines, tissues, and body fluids, etc.) for quantitative proteomic research.
Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China.
Shu-Jun
SJ
Yang
Ai-Ying
AY
Nie
Lei
L
Zhang
Guo-Quan
GQ
Yan
Jun
J
Yao
Li-Qi
LQ
Xie
Hao-Jie
HJ
Lu
Peng-Yuan
PY
Yang
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000737:chemistry; D003903:Deuterium; D006801:Humans; D007553:Isotope Labeling; D008099:Liver / Q000737:chemistry; D008113:Liver Neoplasms / Q000737:chemistry; D008297:Male; D008745:Methylation; D010100:Oxygen; D010103:Oxygen Isotopes; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism; D057188:Workflow
D010103:Oxygen Isotopes; D010446:Peptide Fragments; D003903:Deuterium; D014357:Trypsin; D010100:Oxygen