title: [Elucidating the structure of two cyclotides of Viola tianshanica maxim by MALDI TOF/TOF MS analysis].
abstract: 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.
authors.affiliation: Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Key Laboratory of Biosynthesis of Natural Products, Ministry of Health of PRC & Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education of PRC, Beijing 100050, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Xiang
authors.forename: Guo-Hua
authors.initials: GH
authors.lastname: Du
authors.forename: Xu-Chen
authors.initials: XC
authors.lastname: Wang
authors.forename: Shu-Xiang
authors.initials: SX
authors.lastname: Zhang
authors.forename: Xian-Yun
authors.initials: XY
authors.lastname: Qin
authors.forename: Jian-Qiang
authors.initials: JQ
authors.lastname: Kong
authors.forename: Ke-Di
authors.initials: KD
authors.lastname: Cheng
authors.forename: Yong-Ji
authors.initials: YJ
authors.lastname: Li
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D047169:Cyclotides / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010946:Plants, Medicinal / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D032413:Viola / Q000737:chemistry*
chemical_list: D047169:Cyclotides; C575242:cycloviolacin T1