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Selective Acylation of Primary Amines in Peptides and Proteins

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journal contribution
posted on 07.12.2007, 00:00 by Nicolas Abello, Huib A. M. Kerstjens, Dirkje S. Postma, Rainer Bischoff
N-hydroxysuccinimide (NHS) esters are derivatizing agents that target primary amine groups. However, even a small molar excess of NHS may lead to acylation of hydroxyl-containing amino acids as a side reaction. We report a straightforward method for the selective removal of ester-linked acyl groups after NHS ester-mediated acylation of peptides and proteins. It is based on incubation in a boiling water bath and does not require a change in pH or the addition of chemicals. It is therefore particularly suited for proteomics samples that are often small in volume and contain low amounts of peptides. The method was optimized and evaluated with two peptides and one protein that were acetylated at a high excess of NHS-acetate. While the large molar excess resulted in complete acylation of all primary amines, hydroxyl-containing amino acids were shown to react as well. By incubating the peptide or protein solutions in a boiling water bath, acetyl−ester bonds were hydrolyzed, whereas acetyl−amide bonds remained stable. The reaction was also performed in 6 M guanidine-HCl, which prevented protein precipitation. In conclusion, the present method allows the selective acylation of primary amines by NHS esters and constitutes a valuable alternative to the treatment with hydroxylamine under alkaline conditions.