论文部分内容阅读
为快速准确地分离鉴定天山堇菜的天然环肽分子,采用50%乙醇浸提、不同极性溶剂萃取,利用Sephadex LH-20对含有环肽的正丁醇部分进行色谱分离,然后直接利用DTT还原分离的组分,结合反相高效液相色谱(RP-HPLC)和质谱跟踪确定含有环肽的组分差异洗脱峰,进而分离纯化获得单体分子;利用DTT还原二硫键、碘乙酰胺烷基化保护被还原产生的巯基、蛋白酶Endo-Glu-C、Endo-Lys-C和胰蛋白酶酶解、结合RP-HPLC分离获得肽段;用基质辅助激光解析电离飞行时间质谱(MALDI TOF/TOF MS)进行质谱分析,鉴定了2个环肽分子,其中一个新的环肽分子命名为cycloviolacin T1,另一个为已知结构的varv E。通过此研究建立了一套直接利用DTT还原色谱分离组分、结合MALDI TOF/TOF MS检测进而鉴定天然环肽分子的解析方法。
In order to rapidly and accurately isolate and identify natural cyclic peptides from Viola tianshanica, 50% ethanol and different polar solvents were used for the separation of n-butanol containing cyclic peptides by Sephadex LH-20. The separated components were reduced and identified by reversed-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry followed by separation and purification to obtain monomer molecules. DTT was used to reduce disulfide bonds and iodine-B The amide was alkylated to protect the sulfhydryl group produced by the reduction, protease Endo-Glu-C, Endo-Lys-C and trypsin digestion, and the peptide fragments were obtained by RP-HPLC. Matrix- assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF / TOF MS), two cyclopeptide molecules were identified, one of which was named cycloviolacin T1 and the other was varv E of known structure. Through this study, a direct analytical method was established for the separation of natural cyclopeptide molecules by DTT reduction chromatography combined with MALDI TOF / TOF MS detection.