pH‐regulated formation of side products in the reductive amination approach for differential labeling of peptides in relative quantitative experiments

Abstract

Research Article pH-regulated formation of side products in the reductive amination approach for differential labeling of peptides in relative quantitative experimentsAmong the most common stable-isotope labeling strategies, the reaction of formaldehyde with peptides in the presence of NaCNBH 3 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 could be employed as well without a substantial increase in terms 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 diethylation 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. Keywords:Diethylation / Dimethylation / Quantitative analysis / Reductive amination / Stable isotope labeling DOI 10.1002/elps.201300484Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionOne of the most crucial issues and growing areas of modern proteomics is the relative quantification of the differential expression of proteins in two or more samples representing various conditions of biological systems. The stable isotope labeling techniques developed in the last two decades for relative quantitation experiments space from in vivo metabolic incorporation of heavy isotopes, or isotopically [3][4][5][6]. Many recent reviews give a comprehensive picture of the current tendency for relative quantitative proteomics with stable isotope labeling [7][8][9][10][11].Reductive amination with formaldehyde and NaCNBH 3 is a simple reaction that involves all the free amino groups of peptides, namely N-termini and lysine residues, replacing hydrogens with two methyl groups [12]. A differential isotope labeling can be achieved by employing d(0)-formaldehyde and d(2)-formaldehyde obtaining a mass increment of 28 and 32 Da, respectively, for each derivatized reactive site. Acetaldehyde and d(4)-acetaldehyde are still relatively inexpensive reagents and can be used as diethylating agents, by the same approach, providing a wider separation, in terms of mass units, between the differentially labeled peptides [13].The chemistry of both formaldehyde and acetaldehyde in biological systems features interesting aspects for proteomics investigations. Formaldehyd...

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