Multistep tandem mass spectrometry for sequencing cyclic peptides in an ion-trap mass spectrometer

Collisionally activated decomposition (CAD) of a protonated cyclic peptide produces a superposition spectrum consisting of fragments produced followin g random ring opening of the cyclic peptide to give a set of acylium ions ( or isomeric equivalents) of the same mit. Assignment of the correct sequenc e is often difficult owing to lack of selectivity in the ring opening. A me thod is presented that utilizes multiple stages of CAD experiments in an el ectrospray ion-trap mass spectrometer to sequence cyclic peptides. A primar y acylium ion is selected from the primary product-ion spectrum and subject ed to several stages of CAD. Amino-acid residues are sequentially removed, one at each stage of the CAD, from the C-terminus, until a b(2) ion is reac hed. Results are presented for seven cyclic peptides, ranging in sizes from four to eight amino-acid residues. This method of sequencing cyclic peptid es eliminates ambiguities encountered with other MS/MS approaches. The powe r of the strategy Lies in the capability to execute several stages of CAD u pon a precursor ion and its decomposition products, allowing the cyclic pep tide to be sequenced in an unambiguous, stepwise manner. (C) 1999 American Society for Mass Spectrometry.