CN111896664B - 一种金枪鱼特征胶原肽及在胶原水解物和其制品鉴定中的应用 - Google Patents

一种金枪鱼特征胶原肽及在胶原水解物和其制品鉴定中的应用 Download PDF

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CN111896664B
CN111896664B CN202010330478.6A CN202010330478A CN111896664B CN 111896664 B CN111896664 B CN 111896664B CN 202010330478 A CN202010330478 A CN 202010330478A CN 111896664 B CN111896664 B CN 111896664B
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黄雅钦
郭尚伟
邓贵雅
李雪娟
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Beijing University of Chemical Technology
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Abstract

一种金枪鱼特征胶原肽及在胶原水解物和其制品鉴定中的应用，属于检测技术领域。本发明涉及金枪鱼源性成分的特征胶原肽Gly‑Pro‑Ala‑Gly‑Ala‑Thr‑Gly‑Ser‑Val‑Gly‑Ala‑Hyp‑Gly‑Lys，可通过检测该特征胶原肽，来确定是否含有金枪鱼源性成分。具体地，通过对样品中的蛋白成分进行胰酶酶切，使特征胶原肽游离出来，然后通过液质联用仪检测是否含有该特征胶原肽，来判定是否含有金枪鱼源性成分。该方法具有特征性强，灵敏度高，操作简单，可对胶原水解物及其制品中的金枪鱼源性成分进行测定。

Description

一种金枪鱼特征胶原肽及在胶原水解物和其制品鉴定中的 应用
技术领域
本发明涉及一种金枪鱼特征胶原肽及在胶原水解物和其制品鉴定中的应用，属于医药与食品检测领域。
背景技术
胶原在动物体内含量非常丰富，主要存在于在动物的骨、皮、筋、腱和鳞等结缔组织中。明胶是胶原的降解物，是由动物的骨、皮、筋、腱和鳞等为原料经适度水解而制得，具有优良的理化性能，广泛应用于食品、医药等诸多领域。明胶绝大部分是由猪和牛的皮或骨为原材料，但由于家禽和牲畜的疾病可能带来的安全风险、清真和宗教信仰不同，对明胶进行动物源性成分溯源具有重要意义。
随着人们生活水平的提高，与家禽、牲畜等为原料制备的明胶相比，鱼明胶被认为更安全和健康，其中尤以金枪鱼为原料制备的明胶最为珍贵。明胶中主要成分为胶原蛋白多肽，可利用液质联用仪检测多肽来进行动物源性成分的溯源，然而由于多肽在同一物种中存在突变等现象，选定的多肽能否作为溯源的标记，关系到溯源的成功与否。而对金枪鱼源性成分溯源的特征胶原肽的选择，不但要克服基因突变带来的影响，而且还需在金枪鱼中含有，而其他动物中不含有。选择金枪鱼中不突变的特征胶原肽，成为金枪鱼源性成分溯源的关键。直接采用液质联用仪对样品进行检测分析，无法克服上述困难。从基因入手，分析寻找特征胶原肽，是一条可行的路径。
发明内容
本发明针对上述问题，提供了一种从基因角度得出的金枪鱼特征胶原肽及在胶原水解物和其制品中金枪鱼源性成分的检测方法，该方法操作简单，特征性强，灵敏度高，可用于胶原水解物及其制品中金枪鱼源性成分的溯源鉴定和含量测定。
本发明的技术方案如下：
一种金枪鱼特征胶原肽及在胶原水解物和其制品鉴定中的应用，基于金枪鱼的Ⅰ型胶原蛋白序列中包含特征胶原肽，其氨基酸序列为Gly-Pro-Ala-Gly-Ala-Thr-Gly-Ser-Val-Gly-Ala-Hyp-Gly-Lys，其中Hyp为4羟基脯氨酸或3羟基脯氨酸。它是从牛、猪、马与金枪鱼中COL1A1蛋白的mRNA出发，找出金枪鱼的稳定存在的特征mRNA序列，翻译成蛋白序列后利用液质联用技术检测，最终选定的特征胶原肽。
该特征胶原肽在胶原水解物及其制品鉴定中的应用，包括以下步骤：
(1)待检测样品用胰蛋白酶进行酶切，或提取待检测样品中的蛋白成分用胰蛋白酶进行酶切；检测样品选自胶原水解物及其制品；
(2)放入液质联用仪，并以特征胶原肽或含特征胶原肽的胰蛋白酶酶切后的金枪鱼明胶作为对照品，选择母离子m/z572.1及其子离子进行监测；如果检出该待测样品对应的离子的保留时间与对照品一致，且其子离子与对照品的子离子一致，则所述待测样品含有金枪鱼源性成分，金枪鱼源性成分含量可由对照品与检测样品中的峰面积来计算；如果没有与对照品保留时间一致的该离子，则不含有金枪鱼源性成分。
上述在胶原水解物包括明胶，明胶是由动物的骨、皮和鳞等为原料经水解所制得的明胶，胶原水解物制品是原料中含有明胶的食品、保健品或药品。
采用本发明的方法，能够快速检测胶原水解物及其制品中是否含有金枪鱼源性成分。尽管动物中存在基因突变导致蛋白序列差异，但本发明的特征胶原肽的基因在金枪鱼中保守性强，且为金枪鱼特有，通过该特征胶原肽，可对胶原水解物及其制品中金枪鱼源性成分进行溯源检测。
附图说明
图1是金枪鱼明胶中特征胶原肽的子离子扫描质谱图(母离子m/z572.1，子离子扫描范围m/z200～1300)；
图2是各种动物明胶SRM扫描模式下选择离子对m/z572.1→789.4，917.3监测的质谱图；
图3是以不同含量金枪鱼明胶对应的特征胶原肽的质谱检测峰面积(m/z572.1→789.4)的关系曲线图；
图4是金枪鱼明胶及明胶制品(以QQ糖为例)在SRM扫描模式下选择离子对m/z572.1→789.4，917.3监测的质谱图。
图5是金枪鱼、牛、猪、马的COL1A1蛋白的部分mRNA序列及对应的比较。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1
1材料和试剂
材料：不同动物来源的明胶，分别来源于金枪鱼、牛、猪、马，分别从金枪鱼骨、牛皮、猪皮、马皮提取获得。
试剂：碳酸氢铵(分析纯)、胰蛋白酶(序列纯，购自中国药品生物制品检定研究院)。
2特征胶原肽的筛选与确定
金枪鱼Ⅰ型胶原的COL1A1蛋白具有一定的保守性，我们可以从COL1A1蛋白中筛选出一段肽作为金枪鱼特征胶原肽。为了从基因中筛选出用于检测的稳定的特征胶原肽，应满足以下条件：(a)与该段肽所对应mRNA序列相邻的三个碱基，翻译后应为精氨酸或赖氨酸，且该段肽所对应mRNA序列中第1～3个碱基，翻译后不应为脯氨酸；(b)该段肽所对应的mRNA序列中，碱基C、G的含量应大于60％；(c)该段肽上至少有一个氨基酸对应的mRNA符合以下任一条件：该氨基酸对应的密码子的三个碱基中，若第一个碱基为C或G，则相应地在牛、猪或马中第一个碱基应为G或C；若第二个碱基为A或T，则相应地在牛、猪或马中第二个碱基应为T或A；若第二个碱基为C或T，则相应地在牛、猪或马中第二个碱基应为T或C；(d)利用质谱检测筛选出来的该段肽是否有翻译后修饰(即：是否有脯氨酸发生羟基化)，如果没有翻译后修饰则筛选出来的该段肽即为特征胶原肽；如果有翻译后修饰则翻译后修饰的多肽即为特征胶原肽；(e)通过质谱检测应没有其他干扰信号。为此我们进行了如下实验：
(1)特征胶原肽的初筛
将金枪鱼、牛、猪、马的COL1A1蛋白对应的mRNA序列进行比对，部分序列如图5所示(注：mRNA由细胞核内的DNA转录来的，有时候也用DNA链上的碱基顺序来表示mRNA上的序列)。图5线框中金枪鱼的mRNA序列“GGT CCC GCT GGT GCT ACT GGT TCT GTT GGT GCC CCTGGC AAA”满足上述条件(a)、(b)、(c)；该序列翻译后，对应的多肽为“Gly-Pro-Ala-Gly-Ala-Thr-Gly-Ser-Val-Gly-Ala-Pro-Gly-Lys”，为确定多肽Gly-Pro-Ala-Gly-Ala-Thr-Gly-Ser-Val-Gly-Ala-Pro-Gly-Lys是否为金枪鱼的特征胶原肽。可进一步确定是否有翻译后修饰(即：是否有脯氨酸发生羟基化)，因此进行了下一步的质谱检测试验来确定是否有翻译后修饰。
(2)特征胶原肽的确定
a)取0.05g明胶样品于25ml量瓶中，加少量1％NH4HCO3溶液浸泡10min，60℃加热使其溶解，用1％NH4HCO3溶液稀释至刻度，摇匀，微孔滤膜过滤，精密量取续滤液0.2ml加入2mg/ml的胰蛋白酶溶液20μl，37℃恒温酶解12h。
b)取5μl的金枪鱼明胶的酶解溶液放入液质联用仪检测多肽是否有羟基化修饰。液相条件：C18反相色谱柱(2.1mm×100mm，1.8μm)，流动相A为0.1％甲酸溶液，流动相B为乙腈，流速0.3ml/min；梯度洗脱：0～25min，98％→80％流动相A，2％→20％流动相B。质谱条件：电喷雾正离子模式(ESI+)，分别选择m/z564.1和m/z572.1为母离子进行子离子全扫描。结果：(1)以m/z564.1为母离子的子离子全扫描的质谱图与多肽Gly-Pro-Ala-Gly-Ala-Thr-Gly-Ser-Val-Gly-Ala-Pro-Gly-Lys不符；(2)以m/z572.1为母离子的子离子全扫描的质谱图，该离子为多肽Gly-Pro-Ala-Gly-Ala-Thr-Gly-Ser-Val-Gly-Ala-Hyp-Gly-Lys，见图1。结果表明金枪鱼的mRNA序列“GGT CCC GCT GGT GCT ACT GGT TCT GTT GGT GCC CCTGGC AAA”翻译的肽中有一个氨基酸被修饰了，即该多肽中有一个脯氨酸发生了羟基化，羟基化后的多肽序列为Gly-Pro-Ala-Gly-Ala-Thr-Gly-Ser-Val-Gly-Ala-Hyp-Gly-Lys。
c)取5μl酶解溶液放入液质联用仪检测。液相条件：C18反相色谱柱(2.1mm×100mm，1.8μm)，流动相A为0.1％甲酸溶液，流动相B为乙腈，流速0.3ml/min；梯度洗脱：0～25min，98％→80％流动相A，2％→20％流动相B。质谱条件：电喷雾正离子模式(ESI+)选择进行SRM检测，选择m/z572.1→789.4，917.3作为检测离子对。结果见图2，5.7min处只有金枪鱼明胶中检出相应的离子峰，其他均没有检出。
综上，多肽Gly-Pro-Ala-Gly-Ala-Thr-Gly-Ser-Val-Gly-Ala-Hyp-Gly-Lys可作为金枪鱼特征胶原肽。
实施例2
1材料和试剂
材料：混合明胶样品(包括：含5％金枪鱼明胶的猪明胶、10％金枪鱼明胶的猪明胶、20％金枪鱼明胶的猪明胶、40％金枪鱼明胶的猪明胶、60％鸡明胶的猪明胶，纯金枪鱼明胶)由实施例1中的明胶精确混合制成。
试剂：碳酸氢铵(分析纯)、胰蛋白酶(序列纯，购自中国药品生物制品检定研究院)。
2检测方法
(1)取0.05g明胶样品于25ml量瓶中，加少量1％NH4HCO3溶液浸泡10min，60℃加热使其溶解，用1％NH4HCO3溶液稀释至刻度，摇匀，微孔滤膜过滤，精密量取续滤液0.2ml加入2mg/ml的胰蛋白酶溶液20μl，37℃恒温酶解12h。
(2)取5μl酶解溶液放入液质联用仪检测。液相条件：C18反相色谱柱(2.1mm×100mm，1.8μm)，流动相A为0.1％甲酸溶液，流动相B为乙腈，流速0.3ml/min；梯度洗脱：0～25min，98％→80％流动相A，2％→20％流动相B。质谱条件：电喷雾正离子模式(ESI+)选择进行SRM检测，选择m/z572.1→789.4，917.3作为检测离子对。
以金枪鱼明胶的浓度为横坐标(X)，以峰面积(m/z572.1→789.4)为纵坐标(Y)，进行曲线绘制。结果见图3，标准曲线Y＝105289X-7268，线性相关系数R2＝0.9977，线性良好。可见该法可用于金枪鱼源性成分的含量检测。
实施例3
1材料和试剂
材料：金枪鱼明胶、加入牛明胶的QQ糖(自制)、加入金枪鱼明胶的QQ糖(自制)；
试剂：碳酸氢铵(分析纯)、胰蛋白酶(序列纯，购自中国药品生物制品检定研究院)。
2检测方法
(1)取0.05～0.25g的金枪鱼明胶、加入牛明胶的QQ糖、加入金枪鱼明胶的QQ糖，分别置于25ml量瓶中，加少量1％NH4HCO3溶液浸泡10min，60℃加热使其溶解，用1％NH4HCO3溶液稀释至刻度，摇匀，微孔滤膜过滤，精密量取续滤液0.2ml加入2mg/ml的胰蛋白酶溶液20μl，37℃恒温酶解12h。
(2)取5μl酶解溶液放入液质联用仪检测。液相条件：C18反相色谱柱(2.1mm×100mm，1.8μm)，流动相A为0.1％甲酸溶液，流动相B为乙腈，流速0.3ml/min；梯度洗脱：0～25min，98％→80％流动相A，2％→20％流动相B。质谱条件：电喷雾正离子模式(ESI+)选择进行SRM检测，选择m/z572.1→789.4，917.3作为检测离子对。
结果见图4，5.7min处金枪鱼明胶和加入金枪鱼明胶的QQ糖检出相应的离子峰，加入牛明胶的QQ糖没有检出，可见该法能够特异性检测明胶制品中的金枪鱼源性成分。
Figure BDA0002464214950000061

Claims (5)

1.一种金枪鱼特征胶原肽，其特征在于，其氨基酸序列为Gly-Pro-Ala-Gly-Ala-Thr-Gly-Ser-Val-Gly-Ala-Hyp-Gly-Lys，其中Hyp为4羟基脯氨酸或3羟基脯氨酸。
2.按照权利要求1所述的一种金枪鱼特征胶原肽，其特征在于，所述的特征胶原肽是从牛、猪、马与金枪鱼中COL1A1蛋白的mRNA出发，找出金枪鱼的稳定存在的特征mRNA序列，翻译成蛋白序列后利用液质联用技术检测，最终选定的特征胶原肽。
3.权利要求1所述的一种金枪鱼特征胶原肽的应用，用于胶原水解物及其制品中金枪鱼源性成分的溯源鉴定和含量测定。
4.按照权利要求3所述的应用，其特征在于，包括以下步骤：
(1)待检测样品用胰蛋白酶进行酶切，或提取待检测样品中的蛋白成分用胰蛋白酶进行酶切；检测样品选自胶原水解物及其制品；
(2)放入液质联用仪，并以特征胶原肽或含特征胶原肽的胰蛋白酶酶切后的金枪鱼明胶作为对照品，选择母离子m/z572.1及其子离子进行监测；如果检出该待检 测样品对应的离子的保留时间与对照品一致，且其子离子与对照品的子离子一致，则所述待检 测样品含有金枪鱼源性成分，金枪鱼源性成分含量可由对照品与检测样品中的峰面积来计算；如果没有与对照品保留时间一致的该离子，则不含有金枪鱼源性成分。
5.按照权利要求4所述的应用，其特征在于，上述胶原水解物包括明胶，明胶是由动物的骨、皮和鳞为原料经水解所制得的明胶，胶原水解物制品是原料中含有明胶的食品、保健品或药品。
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