Sequencing cyclic peptide inhibitors of mammalian ribonucleotide reductaseby electrospray ionization mass spectrometry

Mammalian ribonucleotide reductase (mRR) is a potential target for cancer i ntervention. A series of lactam-bridged cyclic peptide inhibitors (1-9) of mRR have been synthesized and tested in previous work. These inhibitors con sist of cyclic and linear regions, causing their mass spectral characteriza tion to be a challenge. We determined the fragmentation mechanism of cyclic peptides 1-9 using an ion-trap mass spectrometer equipped with an ESI sour ce. Low-energy collision-induced dissociation of sodiated cyclic peptides c ontaining linear branches follows a general pathway. Fragmentation of the l inear peptide region produced mainly a and b ions. The ring peptide region was more stable and ring opening required higher collision energy, mainly o ccurring at the amide bond adjacent to the lactam bridge. The sodium ion, w hich bound to the carbonyl oxygen of the lactam bridge, acted as a fixed ch arge site and directed a charge-remote, sequence-specific fragmentation of the ring-opened peptide. Amino acid residues were cleaved sequentially from the C-terminus to the N-terminus. Our findings have established a new way to sequence cyclic peptides containing a lactam bridge based on charge-remo te fragmentation. This methodology will permit unambiguous identification o f high-affinity ligands within cyclic peptide libraries. Copyright (C) 2001 John Wiley & Sons, Ltd.