title: Elucidating the structure of cyclotides by partial acid hydrolysis and LC-MS/MS analysis.
abstract: 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 degrees 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 Asx/Glx and Ser/Thr. In particular, the most susceptible bond for acid hydrolysis in kalata B1 and varv A was found to be the highly conserved N25-G26 which is also the head-to-tail ligation site of the linear precursor proteins, indicating that formation of the three disulfide bonds might precede cyclic structure closure by N25-G26 ligation. This observation is consistent with the recent report that the N25-G26 bond formation is the last step in the cyclotide biosynthetic pathway. The process demonstrated here can potentially be a high throughput method that is generally applicable to determine disulfide bonds of other relatively low-abundance cyclotides.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore. SKSze@ntu.edu.sg
authors.forename: Siu Kwan
authors.initials: SK
authors.lastname: Sze
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Wei
authors.initials: W
authors.lastname: Meng
authors.forename: Randong
authors.initials: R
authors.lastname: Yuan
authors.forename: Tiannan
authors.initials: T
authors.lastname: Guo
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhu
authors.forename: James P
authors.initials: JP
authors.lastname: Tam
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D047169:Cyclotides / Q000737:chemistry*; D006868:Hydrolysis; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D053719:Tandem Mass Spectrometry / Q000379:methods*; D032413:Viola / Q000737:chemistry
chemical_list: D047169:Cyclotides; C091930:kalata B1