Identification of by-products from an orthogonal peptide ligation by oximebonds using mass spectrometry and tandem mass spectrometry

Synthetic proteins with unusual architecture are obtained through chemosele ctive ligation, a method based on the condensation of unprotected peptides under mild aqueous conditions, The last step of a new procedure leading to a tri-branched conjugate consists of the chemoselective ligation reaction b etween an (amioooxy)acetyl peptide and a peptide aldehyde resulting from a first ligation via an oxime bond, In order to optimize the reaction conditi ons, electrospray ionization mass spectrometry combined with Liquid chromat ography and tandem mass spectrometry has been used, In addition to the targ et tri-branched conjugate, two other conjugates were characterized allowing documentation of transoximation reactions in peptide chemistry, A fourth c onjugate was identified as a side product appearing after the first ligatio n, Data obtained by low-energy collision-induced dissociation led to a rapi d and reliable identification of impurities observed in the (aminooxy)acety l peptide despite a previous high performance liquid chromatography (HPLC) purification. This highlights the great reactivity of the aminooxy group to wards carbonyl-containing compounds. Copyright (C) 2000 John Wiley &: Sons, Ltd.