Doubly protonated substance P and two analogs alkylated at the ninth position was studied to determine the effect of N-alkylation of the amide nitrogen on the electrospray ionization/surface-induced dissociation (ESI/SID) fragmentation pattern. Thermal decomposition experiments and ab initio calculations were also used in conjunction with the ESI/SID experiments. The increase in relative abundances of the product ions resulting from the cleavage of the amide bond at the alkylation site (relative to the corresponding cleavage for substance P) can be explained by the increased basicity of the amide nitrogen in the context of the 'mobile proton' model. The relative abundances of singly charged b ions suggest a rearrangement of the amide hydrogen located N-terminal to the bond cleaved.