pH Regulated Formation of Side Products in the Reductive Amination Approach for Differential Labeling of Peptides in Relative Quantitative Experiments.

Among the most common stable-isotope labeling strategy, the reaction of formaldehyde with peptides in the presence of NaCNBH3 features many attractive aspects that are conducive to its employment in quantitation experiments in proteomics. Reductive amination, with formaldehyde and d(2)-formaldehyde, is reported to be a fast, easy, and specific reaction, undoubtedly inexpensive if compared with commercially available kits for differential isotope coding. Acetaldehyde and d(4)-acetaldehyde have been recently employed as well to obtain , could be employed as well without a substantial increase of cost, and should provide a wider spacing between the differentially tagged peptides in the mass spectrum. Nevertheless only a single paper reports about a di-ethylation approach for quantitation. We undertook a systematic analytical investigation on the reductive amination of some standard peptides pointing out the occasional occurrence of side reactions in dependence of pH or reagents order of addition, particularly observing the formation of cyclic adducts ascribable to rearrangements involving the generated Schiff-base, and all the nucleophilic sites of its chemical environment. We also tried to evaluate how much this side-products amount may impair isotope coded relative quantitation.