Non-gel-based dual ¹⁸O labeling quantitative proteomics strategy

Abstract

To improve the quantitation of target proteins in proteomic analyses, we developed a non-gel-based, dual 18O 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 18O labeling method (chemical 18O labeling) of our dual strategy, one functional group was covalenuy coupled to the primary amines of the peptides and 18O from H218O was incorporated at the other functional group by hydrolysis. In the second 18O labeling method (chemical and enzyme-catalyzed 18O labeling), chemical 18O labeling and enzyme-catalyzed 18O 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 18O labeling were accurate in the range 0.1-10 of 16O/18O peptide ratios. The deviations of the methods were <20%. In contrast to current proteolytic 18O labeling methods, there was no 18O to 16O back-exchange in the first method and no isotope peaks in MS in the second method. The combination of chemical and proteolytic 18O labeling improved the confidence of the quantitation results. © 2007 American Chemical Society.