Elucidating the structure of two cyclotides of Viola tianshanica Maxim by MALDI TOF/TOF MS analysis

The cyclotides are a family of cyclic “mini” proteins that occur in Violaceae, Rubiaceae and   Cucurbitaceae plant families and contain a head-to-tail cyclic backbone and a cystine knot arranged by three   disulfide bonds.  To study the natural cyclotides of V. tianshanica, dried herb was extracted with 50% ethanol, and the concentrated aqueous extract was subjected to a solvent-solvent partitioning between water and hexane, ethyl acetate and n-butanol, separately.  The n-butanol extract containing cyclotides was subjected to column chromatography over Sephadex LH-20, eluted with 30% methanol.  The subfractions were directly reduced by DTT and analyzed by reverse-phase HPLC.  The peaks with different retention times were shown on the profile of RP-HPLC and collected.  The cyclotides were speculated based on masses range from 3 000 to 3 500 Da.  The purified cyclotides were reduced with DTT, alkylated with iodoacetamide, and then were cleaved with  endoproteinase Glu-C, endoproteinase Lys-C and Trypsin, separately.  The digested peptides were purified on RP-HPLC and analyzed on MALDI TOF/TOF analyzer.  A new cyclotide, cycloviolacin T1 and a reported cyclotide varv E were systemically determined using MALDI TOF/TOF system.  So the method for the isolation and characterization of cyclotides was quickly built up in succession.