首页|半夏炮制过程中毒性凝集素蛋白的基质辅助激光解吸电离飞行时间质谱分析

半夏炮制过程中毒性凝集素蛋白的基质辅助激光解吸电离飞行时间质谱分析

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基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)受灵敏度和重复性的影响,对完整蛋白的定量分析存在困难.本研究采用纤维素纳米晶体(CNC)辅助MALDI-TOF MS,结合内标法定量分析半夏炮制过程中半夏凝集素(PTL),通过优化CNC辅助样本制备的时长和比例,以麦胚凝集素(TVL)为内标,直接分析不同炮制程度半夏各炮制品及其浸泡上清液中PTL含量.结果表明,CNC辅助MALDI样本制备有效提升了 MALDI-TOF MS定量分析的线性和重复性.随着炮制时间的延长,姜半夏、清半夏和法半夏炮制品的PTL含量逐渐下降,浸泡上清液中PTL含量逐渐上升.其中,由于pH值的改变,法半夏炮制品浸泡上清液中PTL含量呈先上升后下降的趋势.此外,将该方法应用于不同饮片生产企业的多批次半夏炮制品饮片中,均未检出PTL.该方法操作简便,可有效分析半夏炮制过程中PTL含量的变化,为研究半夏炮制过程对PTL的影响提供了参考.
Analysis of Pinellia ternata Toxic Lectin Protein During Processing by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
Pinellia ternata lectin(PTL)is considered as the toxic substance of Pinellia ternata,and is the main cause of irritation in raw Pinellia tubers.The content of PTL decreases after processing.Pinellia tubers are processed with ginger extract,liquorice or alum to reduce the toxicity and get processed products,such as Jingbanxia,Qingbanxia and Fabanxia.Matrix-assisted laser desorption/ionization time-of-flight mass spectrom-etry(MALDI-TOF MS)method has the advantages of low cost,rapid and automated operation,which is suitable for the detection of intact proteins.However,MALDI-TOF MS is difficult to perform quantitative analysis of intact protein due to its low sensitivity and repeatability.It is important to establish a rapid analysis method for the detection of PTL by improving the sensitivity and repeatability of MALDI-TOF MS.Cellulose nano-crystal(CNC)is one of the nano materials with rod-like structure,which has wide applications due to its large specific surface area,abundant hydroxyl groups.It has the ability to reduce protein aggregation,so can reduce the aggregated protein ion[nM+H]+signals in MALDI-TOF MS.In this study,CNC assisted MALDI-TOF MS combined with internal standard method was used to quantitative analyze PTL during processing of Pinellia ternata.The preparation time and proportion of CNC-assisted samples were optimized,and Triticum vulgaris lectin(TVL)was used as internal standard to directly analyze PTL in processed products and their supernatant.Good repeatability of MALDI-TOF MS analysis could be successfully achieved by adding CNC to the protein sample solution.CNC-assisted MALDI sample preparation could effec-tively solve the quantitative analysis problem of MALDI-TOF MS,improve the sensitiv-ity,linearity and repeatability.During the processing of raw Pinellia tubers,the PTL in raw tubers was gradually dissolved in the Pinellia tubers extract.As the processing time increased,the content of PTL in the three types of processed products gradually decreased,and gradually increased in their supernatant.The content of PTL in the soak supernatant tended to increase and then decrease due to pH change.In addition,PTL was not detected in many batches of processed products from different manufacturers.Instead,PTL was detected at high levels in raw Pinellia tubers.The method provides a scientific basis for the mechanism of Pinellia ternata processing on toxic PTL,as well as for the optimization of processing production and technological innovation.

Pinellia ternataprocessing methodPinellia ternata lectin(PTL)cellu-lose nanocrystals(CNC)matrix assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS)

黄益曼、李春晖、柏家辉、陆颖洁、李添、董志颖、苏越、郭寅龙

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上海中医药大学交叉科学研究院,上海 201203

中国科学院上海有机化学研究所,金属有机化学国家重点实验室,上海 200032

上海中医药大学中药学院,上海 201203

半夏 炮制方法 半夏凝集素(PTL) 纤维素纳米晶体(CNC) 基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)

国家重点研发计划上海市科技创新行动计划上海市科技创新行动计划中医药流派发展高地建设海派中药炮制技术传承创新平台建设项目

2021YFF07019002114220080022DZ2204300ZY2021-2023-0210

2024

10.7538/zpxb.2023.0133

质谱学报
北京中科科仪技术发展有限责任公司 中国物理学会质谱分会

质谱学报

CSTPCD北大核心EI
影响因子：1.046
ISSN：1004-2997
年,卷(期)：2024.45(2)
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