Non-gel-based dual 18O labeling quantitative proteomics strategy.
To improve the quantitation of target proteins in proteomic analyses, we developed a non-gel-based, dual (18)O labeling strategy. This global isotope labeling method utilizes an acylating chemical reagent with two anhydride functional groups, bicyclic anhydride diethylenetriamine-N,N,N', N' ',N' '-pentaacetic acid (DTPA) dianhydride. In the first (18)O labeling method (chemical (18)O labeling) of our dual strategy, one functional group was covalently coupled to the primary amines of the peptides and (18)O from H2(18)O was incorporated at the other functional group by hydrolysis. In the second (18)O labeling method (chemical and enzyme-catalyzed (18)O labeling), chemical (18)O labeling and enzyme-catalyzed (18)O labeling of the carboxyl- termini of the peptides were combined. The acylation reaction between DTPA and the model peptides was rapid and specific, and the DTPA-modified N-termini of the peptides promoted only y-series ions in MS/MS. The two methods of (18)O labeling were accurate in the range 0.1-10 of (16)O/(18)O peptide ratios. The deviations of the methods were <20%. In contrast to current proteolytic (18)O labeling methods, there was no (18)O to (16)O back-exchange in the first method and no isotope peaks in MS in the second method. The combination of chemical and proteolytic (18)O labeling improved the confidence of the quantitation results.
Liu
Huiling
H
State Key Lab of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, 33 Zhongguancun Life Park Road, Beijing 102206, China.
Zhang
Yangjun
Y
Meng
Lingyan
L
Qin
Peibing
P
Wei
Junying
J
Jia
Wei
W
Li
Xiaofeng
X
Cai
Yun
Y
Qian
Xiaohong
X
D000588:Amines / Q000737:chemistry; D005782:Gels / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling; D015394:Molecular Structure; D010103:Oxygen Isotopes / Q000737:chemistry; D004369:Pentetic Acid; D010455:Peptides / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000588:Amines; D005782:Gels; D010103:Oxygen Isotopes; D010455:Peptides; D004369:Pentetic Acid