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The fragmentation characteristics of peptides derivatized at the side-chain epsilon-amino group of lysyl residues via reductive amination with benzaldehyde have been examined using collision-induced dissociation (CID) tandem mass spectrometry. The resulting MS/MS spectra exhibit peaks representing product ions formed from two independent fragmentation pathways. One pathway results in backbone fragmentation and commonly observed sequence ion peaks. The other pathway corresponds to the unsymmetrical, heterolytic cleavage of the C(zeta)-N(epsilon) bond that links the benzyl derivative to the side-chain lysyl residue. This results in the elimination of the derivative as a benzylic or tropylium carbocation and a (n - 1)(+)-charged peptide product (where n is the precursor ion charge state). The frequency of occurrence of the elimination pathway increases with increasing charge of the precursor ion. For the benzyl-modified tryptic peptides analyzed in this study, peaks representing products from both of these pathways are observed in the MS/MS spectra of doubly-charged precursor ions, but the carbocation elimination pathway occurs almost exclusively for triply-charged precursor ions. The experimental evidence presented herein, combined with molecular orbital calculations, suggests that the elimination pathway is a charge-directed reaction contingent upon protonation of the secondary epsilon-amino group of the benzyl-derivatized lysyl side chain. If the secondary epsilon-amine is protonated, the elimination of the carbocation is observed. If the precursor is not protonated at the secondary epsilon-amine, backbone fragmentation persists. The application of appropriately substituted benzyl analogs may allow for selective control over the relative abundance of product ions generated from the two pathways., (2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.)