Electrospray ionization/collision-induced dissociation of protonated substance P ions--effect of charge on the fragmentation patterns

Low-energy collision-induced dissociation (CID) of singly charged [M + H]+ and doubly charged [M + 2H]2+ substance P (SP) ions generated by electrospray ionization (ESI) was studied using a Micromass Q-TOF2 MS instrument. SP (RPKPQQFFGLM-NH2) is a naturally occurring 11 amino acid residue C-terminally amidated peptide, a potent modulator of neuroimmunoregulation. Its N-terminal is the most basic amino acid residue--arginine. Upon CID, both the singly charged and doubly charged SP ions produced primarily N-terminal fragment ions (a and b ions, and their neutral loss a-17 and b-17 ions). However, the relative abundance of these ions showed significant difference: Singly charged SP showed dominant b, a-17 and b-17 ions, while doubly charged SP dominant a and b ions. The complete sequence of substance P can be obtained by a combination of the CID data of the two SP ions. The singly charged and doubly charged SP ions also yielded several prominent internal fragment ions, the relative abundance of which also depended on the charge status. These results provide insights into the CID fragmentation mechanisms of the two SP ions.