摘要
由于角蛋白结构稳定,富含角蛋白的角类动物药提取溶解步骤复杂,严重制约了检测效率及通量,该文通过简化角类样品前处理步骤,优化主要角类动物药的特征肽检测方法,提升角类动物药专属性鉴别效率。分别对经/未经碘乙酰胺(IAA)处理的角类动物药特征肽类进行分析,优化特征肽的离子对条件,并比较经/未经IAA处理的特征肽的保留时间、响应值等数据;采用直接酶解与提取总蛋白后酶解2种前处理方法制备角类动物药样品,基于特征肽保留时间、离子响应值等比较不同方法对羚羊角、水牛角、山羊角及牦牛角样品中特征肽检测的影响。结果表明待测角类样品经直接酶解的简化工艺处理后,均可检测到未经IAA处理的特征肽信息,且与IAA处理后的特征肽相比,其保留时间后移,质谱响应稍有降低;未经IAA处理的特征肽专属性良好,不影响角类动物药的专属性鉴别。综上,可采用直接酶解的工艺处理角类样品,省略蛋白质提取、二硫苏糖醇、IAA试剂处理步骤,显著提高前处理效率,且不影响角类动物药的专属性鉴别,为角类动物药质量研究及标准提升提供了思路与策略。
Due to the highly stable structure of keratin,the extraction and dissolution steps of animal medicines rich in keratin are complex,which seriously restricts the detection efficiency and flux.Therefore,this study simplified the pre-treatment steps of horn samples and optimized the detection methods of characteristic peptides to improve the efficiency of identifying the specificity of horn-de-rived animal medicines.For detection of the characteristic peptides in horn-derived animal medicines treated with/without iodoace-tamide(IAA),the ion pair conditions of the characteristic peptides were optimized,and the retention time,intensity and other data of the specific peptides were compared between the samples treated with/without IAA.Two pre-treatment methods,direct enzymatic hydrolysis and total protein extraction followed by enzymatic hydrolysis,were used to prepare horn-derived animal medicine samples.The effects of different methods on the detection of specific peptides in the samples of Saiga antelope horn,water buffalo horn,goat horn,and yak horn were compared regarding the retention time of specific peptides and ion intensity.The results indicated that after direct enzymatic hydrolysis,the specific peptides in the samples without IAA treatment can be detected.Compared with the characteristic peptides in the samples treated with IAA,their retention time shifted back and the mass spectrometry response slightly decreased.The specific peptides of the samples without IAA treatment had good specificity and did not affect the specificity identification of horn-derived animal medicines.Overall,the process of direct enzymatic hydrolysis can be used to treat horn samples,omitting the steps of protein extraction and dithiothreitol and IAA treatment,significantly improving the pre-treatment efficiency without affecting the specificity identification of horn-derived animal medicines.This study provides ideas for quality research and standard improvement of horn-derived animal medicines.
作者
黄潇正
武文星
黄思莹
包旺林
冯旗园
赵明
段金廒
刘睿
HUANG Xiao-zheng;WU Wen-xing;HUANG Si-ying;BAO Wang-lin;FENG Qi-yuan;ZHAO Ming;DUAN Jin-ao;LIU Rui(National Administration of Traditional Chinese Medicine Key Laboratory of Chinese Medicinal Resources Recycling Utilization,National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine,Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization,Nanjing University of Chinese Medicine,Nanjing 210023,China;Animal-Derived Chinese Medicine and Functional Peptides International Collaboration Joint Laboratory,Nanjing 210023,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2024年第9期2402-2409,共8页
China Journal of Chinese Materia Medica
基金
国家自然科学基金面上项目(81973450)
国家重点研发计划项目(2018YFC1706100)
江苏省高校“青蓝工程”中青年学术带头人项目
江苏省农业科技自主创新项目(CX(22)3173)
南京中医药大学中药学一流学科开放课题(2020YLXK009)。
