[关键词]
[摘要]
目的 基于蛋白质、肽类物质组成及修饰组成的变化,研究砂炒炮制对穿山甲物质基础的影响。方法 采用Nano LC-MS/MS对炮制前后穿山甲蛋白质、肽类组成及变化进行分析鉴定,对鉴定的蛋白质、肽类发生的脱酰胺修饰与氧化修饰进行比较。结果 穿山甲主要由角蛋白、连接蛋白、桥粒蛋白等结构蛋白及促进角质致密结构形成的异构酶类组成。穿山甲经炮制后,可溶性蛋白质、肽类的鉴定数量未见明显变化,难溶性蛋白质鉴定数量显著降低;炮制后蛋白质、肽类的脱酰胺修饰数量显著增加;炮制后I型、Ⅱ型角蛋白的鉴定肽段数量显著增加,Asn与Gln发生脱酰胺修饰的数量显著增加;穿山甲炮制前后鉴定肽段的相对分子质量分布及亲疏水性无显著变化。结论 尽管炮制会降低穿山甲整体蛋白质鉴定的数量,但可显著增加角蛋白的鉴定肽段数量,可显著提高蛋白质、肽类成分的脱酰胺修饰数量。结果提示炮制过程有利于角蛋白等结构蛋白的蛋白质、肽类成分的溶出与释放。为穿山甲物质基础研究,炮制对角质类动物药物质基础的影响研究等提供依据,也为穿山甲替代资源寻找与评价提供研究思路与方法。
[Key word]
[Abstract]
Objective To study the effect of high temperature sand fried processing on the chemical constituents in Manis Squama based on the changes of proteins, peptides, and modifications. Methods Nano LC-MS/MS was used to analyze and identify proteins and peptides in Manis Squama before and after processing. The PTMs including deamidation and oxidation occurred in Manis Squama during processing were also investigated. Results The results showed that Manis Squama consisted of keratins, connexin, desmoplakin, and some isomerases, which can promote the keratinous structure formation. After processing, the number of identified proteins and peptides in soluble fraction did not show significant change, while the number of proteins and peptides in in-soluble faction were decreased significantly. The number of deamidation was increased significantly, and the number of deamidation on Asn and Gln in Keratins was increased significantly. There were no significantly change on molecular weight distribution and GRAVY value. Conclusion High temperature sand fried processing can decrease the protein and peptide identifications of Manis Squama, but significantly increase the identifications from Keratins and other structural proteins, and significantly increase the number of deamidation, which might help to increase the releasing and dissolution of soluble proteins and peptides. In this study, we provided important evidences on revealing the effect of processing on the chemical constituents changing in Manis Squama, also provided ideas and methods for searching and evaluation of Manis Squama alternative resources.
[中图分类号]
R283.1
[基金项目]
国家重点研发计划资助(2018YFC1706100);国家自然科学基金面上项目(81973450);江苏省高校“青蓝工程”优秀青年骨干教师(2016年);江苏省“333工程”第三层次(2016年);中华中医药学会“青年人才托举工程”(QNRC2-C14);江苏省高等学校自然科学研究项目(19KJB360020)