Elucidating the structure of two cyclotides of Viola tianshanica maxim by MALDI TOF/TOF MS analysis.
- Author:
Bin XIANG
1
;
Guo-Hua DU
;
Xu-Chen WANG
;
Shu-Xiang ZHANG
;
Xian-Yun QIN
;
Jian-Qiang KONG
;
Ke-Di CHENG
;
Yong-Ji LI
;
Wei WANG
Author Information
1. 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.
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
Chromatography, High Pressure Liquid;
Cyclotides;
chemistry;
isolation & purification;
Molecular Sequence Data;
Molecular Structure;
Plants, Medicinal;
chemistry;
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization;
Tandem Mass Spectrometry;
Viola;
chemistry
- From:
Acta Pharmaceutica Sinica
2010;45(11):1402-1409
- CountryChina
- Language:Chinese
-
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.
