The complexes formed by alkali metal cations (Cat+ = Li+, Na+, K+, Rb+) and singly charged tryptic peptides were investigated by combining results from the low-energy collision-induced dissociation (CID) and ion mobility experiments with molecular dynamics and density functional theory calculations. The structure and reactivity of [M + H + Cat]2+ tryptic peptides is greatly influenced by charge repulsion as well as the ability of the peptide to solvate charge points. Charge separation between fragment ions occurs upon dissociation, i.e. b ions tend to be alkali metal cationised while y ions are protonated, suggesting the location of the cation towards the peptide N-terminus. The low-energy dissociation channels were found to be strongly dependant on the cation size. Complexes containing smaller cations (Li+ or Na+) dissociate predominantly by sequence-specific cleavages, whereas the main process for complexes containing larger cations (Rb+) is cation expulsion and formation of [M + H]+. The obtained structural data might suggest a relationship between the peptide primary structure and the nature of the cation coordination shell. Peptides with a significant number of side chain carbonyl oxygens provide good charge solvation without the need for involving peptide bond carbonyl groups and thus forming a tight globular structure. However, due to the lack of the conformational flexibility which would allow effective solvation of both charges (the cation and the proton) peptides with seven or less amino acids are unable to form sufficiently abundant [M + H + Cat]2+ ion. Finally, the fact that [M + H + Cat]2+ peptides dissociate similarly as [M + H]+ (via sequence-specific cleavages, however, with the additional formation of alkali metal cationised b ions) offers a way for generating the low-energy CID spectra of ‘singly charged’ tryptic peptides. Copyright © 2010 John Wiley & Sons, Ltd.