AbstractWe have examined the multi‐stage collision induced dissociation (CID) of metal cationized leucine enkephalin, leucine enkephalin amide, and the N‐acetylated versions of the peptides using ion trap mass spectrometry. In accord with earlier studies, the most prominent species observed during the multi‐stage CID of alkali metal cationized leucine enkephalin are the [bn + 17 + Cat]+ ions. At higher CID stages (i.e. >MS4), however, dissociation of the [b2 + 17 + Cat]+ ion, a cationized dipeptide, results in the production of [an − 1 + Cat]+ species. The multi‐stage CID of Ag+ cationized leucine enkephalin can be initiated with either the [bn − 1 + Ag]+ or [bn + 17 + Ag]+ ions produced at the MS/MS stage. For the former, sequential CID stages cause, in general, the loss of CO, and then the loss of the imine of the C‐terminal amino acid, to reveal the amino acid sequence. Similar to the alkali cationized species, CID of [b2 − 1 + Ag]+ produces prominent [an − 1 + Ag]+ ions. The multi‐stage CID of argentinated peptides is reminiscent of fragmentation observed for protonated peptides, in that a series of (bn) and (an) type ions are generated in sequential CID stages. The Ag+ cation is similar to the alkali metals, however, in that the [bn + 17 + Ag]+ product is produced at the MS/MS and MS3 stages, and that sequential CID stages cause the elimination of amino acid residues primarily from the C‐terminus. We found that N‐acetylation of the peptide significantly influenced the fragmentation pathways observed, in particular by promoting the formation of more easily interpreted (in the context of unambiguous sequence determination) dissociation spectra from the [b2 + 17 + Li]+, [b2 + 17 + Na]+ and [b2 − 1 + Ag]+ precursor ions. Our results suggest, therefore, that N‐acetylation may improve the efficacy of multi‐stage CID experiments for C‐terminal peptide sequencing in the gas phase. For leucine enkephalin amide, only the multi‐stage CID of the argentinated peptide allowed the complete amino acid sequence to be determined from the C‐terminal side. Copyright © 2002 John Wiley & Sons, Ltd.