纳升液相色谱-静电场轨道阱质谱法检测鹅膏菌属毒性相关内源性前体蛋白

Determination of toxic related endogenous precursor proteins of Amanita by nanolitron liquid chromatography-electrostatic field orbital trap mass spectrometry

中文关键词:  #$TAB：纳升液相色谱-静电场轨道阱质谱法  MSDIN毒性相关内源性前体蛋白  鹅膏菌属

英文关键词:nanolitron liquid chromatography-electrostatic field orbital trap mass spectrometry  MSDIN toxic related endogenous precursor proteins  Amanita

中文摘要:

      目的 建立纳升液相色谱-静电场轨道阱质谱法测定鹅膏菌属中MSDIN毒性相关前体蛋白的方法。方法 样品经纯水提取、超滤分离后, 采用StageTips C18进行样品除盐; 以0.1%甲酸水溶液和含0.1%甲酸的乙腈/水(4:1, V:V)溶液为流动相, 采用梯度洗脱方式, 经Acclaim? PepMap? 100 C18 (70 mm×75 μm, 3 μm)预富集, Acclaim? PepMap? 100 C18 (150 mm×75 μm, 3 μm)分离, 采用电喷雾离子源正离子模式测定, Protein Discover软件对蛋白质进行鉴定; 分别对蛋白提取液种类、蛋白质纯化方法和蛋白酶种类进行对比和优化。结果 优化前处理条件后选择纯水作为提取溶剂, 提取液经Vivaspin Turbo 4 (3 kDa)分离纯化和StageTips C18除盐后, 样品在非酶解状态下进行分析; 将本方法用于2017—2020年四川地区食物中毒事件中收集到的23株鹅膏菌属类蘑菇, 共鉴定MSDIN毒性相关内源性前体蛋白50个; 与市售8种可食用蘑菇比较, 差异表达前体蛋白41个, 其中13条前体蛋白为全部23株鹅膏菌属类蘑菇共有, 可作为鹅膏菌属潜在的特征性靶标分子。结论 该方法简单快速、特异性强, 可作为形态学鉴定、DNA测序和毒素检测的补充方法, 初步用于鹅膏菌属的快速识别。

英文摘要:

      Objective To establish a method for the determination of MSDIN genes coded, endogenous toxic proproteins in Amanita genus by nanolitron liquid chromatography-electrostatic field orbital trap mass spectrometry. Methods After the water extraction and ultrafiltration, the sample was desalted by StageTips C18. A gradient elution program was used with a 0.1% formic acid aqueous solution and a mixture of acetonitrile/water (4:1, V:V) containing 0.1% formic acid as the mobile phase. Proteins, which were first bonded in an Acclaim? PepMap?100 C18 (70 mm×75 μm, 3 μm) pre-enrichment column, then separated on an Acclaim? PepMap?100 C18 (150 mm×75 μ m, 3 μm) column. The ionization mode was set as positive ion mode of electrospray ion source. Protein Discover software was applied for protein identification. The types of protein extract, methods of protein purification and types of protease were compared and optimized. Results After optimizing the pre-treatment conditions, set water was used as the extraction solvent, the extract was then separated and purified by Vivaspin Turbo 4 (3 kDa) and desalinated by StageTips C18. The sample was analyzed in a non enzymatic condition. A total of 50 MSDIN related proproteins were identified in 23 Amanita strains that collected in food poisoning incidents in Sichuan from 2017 to 2020. Compared with proteins found in 8 edible mushrooms, 41 proproteins were identified as Amanita specificity. Among which 13 proproteins were found in all 23 test strains, and could serve as the potential characteristic target molecules. Conclusion The method is capable of simplicity, velocity and specificity, and can be preliminarily applied for rapid identification of Amanita as complementary methods for morphological identification, DNA sequencing and toxins detection.