Matrix-assisted laser desorption/ionisation (MALDI) mechanisms and the factors that influence the intensity of the ion signal in the mass spectrum remain imperfectly understood. In proteomics, it is often necessary to maximise the peptide response in the mass spectrum, especially for low abundant proteins or for proteolytic peptides of particular significance. We set out to determine which of the common proteolytic enzymes give rise to peptides with the best response factors under MALDI conditions. Standard proteins were enzymatically digested using four common proteases. We assessed relative response factors by coanalyzing the resulting digests. Thus, when tryptic peptides were added in equimolar quantities to their corresponding Asp-N, chymotrypsin and Glu-C digests, tryptic peptide signals were always predominant in the resulting MALDI mass spectra. Observable peaks attributable to non-tryptic peptides generally contained a terminal basic residue. It was proposed that a terminal basic residue has a disproportionate influence upon gas-phase basicity, and this hypothesis was supported by experiments with model isotopically labelled peptides. Experiments applying Cook's kinetic method showed that the peptide with a C-terminal arginine residue was more basic than the equivalent peptide with an N-terminal arginine, which was more basic than the peptide in which the arginine was mid-chain. Thus, the observation of the higher MALDI mass spectrometry response factors of tryptic peptides in comparison with peptides derived using other proteolytic enzymes corresponds with higher gas-phase basicities and may, along with other factors such as the complexity of the digest, influence the choice of enzyme in “bottom–up” proteomic experiments. Copyright © 2011 John Wiley & Sons, Ltd.