Elucidating the structure of cyclotides by partial acid hydrolysis and LC-MS/MS analysis.

We describe here a rapid method to determine the cyclic structure and disulfide linkages of highly stable cyclotides via a combination of flash partial acid hydrolysis, LC−MS/MS, and computational tools. Briefly, a mixture of closely related cyclotides, kalata B1 and varv A purified from Viola yedoensis was partially hydrolyzed in 2 M HCl for 5 min by microwave-assisted hydrolysis or for 30 min in an autoclave oven (121 °C and 15 psi). The partially hydrolyzed peptide mixture was then subjected to LC−MS/MS analysis, with the disulfide linked-peptides fragmented by collision activated dissociation (CAD). A computer program written in-house (available for download at http://proteomics.sbs.ntu.edu.sg/cyclotide_SS) was used for interpreting LC−MS/MS spectra and assigning the disulfide bonds. Time-point analysis of single-disulfide fragments revealed that nonrandom acid catalyzed fragmentation mostly occurred at the turns which are solvent-exposed and often contain side chain functionalized amino acids such as...