Mass Spectrometric Analysis of Cyclotides from Clitoria ternatea: Xxx-Pro Bond Fragmentation as Convenient Diagnostic of Pro Residue Positioning

Cyclotides, a class of macrocyclic plant peptides, characterized by a cyclic backbone and three inter-locking disulfide bonds, may be divided into two major structural subfamilies, Mobius and Bracelet, based on the presence or absence of a specific proline residue. The present study describes the suite of cyclotides obtained from Clitoria ternatea, characterized by LC-MS and MS/MS techniques. Notable variations in product ion distributions were observed in cyclotides belonging to different structural subfamilies based on the number and positions of proline residues. For instance, Cter M which is an abundant Mobius cyclotide in this plant containing three proline residues, displayed distinct b- and y- ion characteristics in the MS/MS spectra compared to Cliotide T1, another commonly identified cyclotide but belonging to the Bracelet subfamily having two proline residues. The distinct fragmentation pattern of prototypical cyclotides of each structural subfamily, determined by Xxx-Pro bond fragmentation, was used to rapidly identify and sequence a novel cyclotide ctr pep 30 from this plant.

Automated summary

Cyclotides, a class of macrocyclic plant peptides, were analyzed from Clitoria ternatea using LC-MS and MS/MS techniques. Variations in product ion distributions were observed in cyclotides belonging to different structural subfamilies based on the number and positions of proline residues. The distinct fragmentation pattern of prototypical cyclotides of each structural subfamily was used to rapidly identify and sequence a novel cyclotide ctr pep 30 from this plant.