马皮特征肽的发现及其在阿胶中马皮源成分检测中的应用

目的：发现马皮特征肽，建立阿胶中马皮源成分专属性的检测方法。方法：采用序列比对技术发现理论马皮胶原蛋白特征肽，利用胰蛋白酶酶切马皮胶样品，通过纳升液相-高分辨质谱确证了马皮特征肽GASGPAGVR。利用超高效液相色谱-三重四极杆质谱（UPLC-QQQ/MS）对马皮特征肽进行检测。色谱柱为ACQUITY UPLC BEH C₁₈（2.1 mm×150 mm，1.7 μm），流动相为0.1%甲酸溶液（A）-乙腈（B），梯度洗脱（0~3 min，96% A；3~8 min，96% A → 92% A；8~10 min，92% A → 50% A；10~12 min，50% A；12~12.1 min，50% A → 96% A；12.1~15 min，96% A），流速0.3 mL·min^-1，柱温40℃，进样量5 μL；离子化模式为ESI⁺，进行多反应监测，选择马皮特征肽特征分子离子峰m/z 386.25→499.25，m/z 386.25→556.51作为检测离子对。结果：在马皮胶样品中检出了马皮特征肽，在阿胶、牛皮胶、猪皮胶、羊皮胶样品中均未检出马皮特征肽。该方法的检出限度为1%马皮胶的阿胶样品。对10批阿胶样品进行检测，在2批样品中检出了马皮特征肽。结论：所建立的方法经验证，专属性强，可用于阿胶中马皮源成分的检测。

Objective:To find marker peptide of horse hide and establish a specific method for identification of horse-hide gelatin in donkey-hide gelatin.Methods:Theoretical species-specific peptide of horse skin was predicted by sequence comparison as GASGPAGVR,which was verified by Nano LC-Orbitrap MS followed by sample digestion with trypsin.Ultra-high performance liquid chromatography coupled with triple quadruple mass spectrometry(UPLC-QQQ/MS)was used to detect horse-hide marker peptide.Determination was carried out on an ACQUITY UPLC BEH C₁₈(2.1 mm×150 mm,1.7 μm)column at temperature of 40℃.The mobile phase was composed of 0.1% formic acid(A)and acetonitrile(B)with gradient elution(0-3 min,96%A;3-8 min,96%A → 92%A;8-10 min,92%A →50%A;10-12 min,50%A;12-12.1 min,50%A → 96%A;12.1-15 min,96%A)at a flow rate of 0.3 mL·min^-1.The electrospray ionization(ESI)source was performed in multiple reaction monitoring(MRM)mode with the transitions of m/z 386.25→499.25 and m/z 386.25→556.51.Results:Marker peptide was found in horse hide gelatin,but not in donkey hide gelatin,cow hide gelatin,pig hide gelatin nor sheep hide gelatin.The detection limit is 1% horse hide gelatin in donkey hide gelatin.Marker peptide was detected in 2 out of 10 donkey-hide gelatin samples.Conclusion:The established method is proved to be specific,which is suitable for the identification of horse-hide gelatin in donkey-hide gelatin.