CN111896663B - 鸡源的特征性ⅲ型胶原肽及在胶原水解物和其制品检测中的应用 - Google Patents
鸡源的特征性ⅲ型胶原肽及在胶原水解物和其制品检测中的应用 Download PDFInfo
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Abstract
鸡源的特征性Ⅲ型胶原肽及在胶原水解物和其制品检测中的应用，属于检测技术领域。鸡源性成分的特征性Ⅲ型胶原肽氨基酸序列Gly‑Ala‑Gln‑Gly‑Pro‑Pro‑Gly‑Pro‑Thr‑Gly‑Ala‑Arg和Gly‑Ser‑Hyp‑Gly‑Pro‑Hyp‑Gly‑Pro‑Ser‑Gly‑Pro‑Ala‑Gly‑Asp‑Arg，通过检测其中一条或两条肽，确定是否含有鸡源性成分。通过对样品中的蛋白成分进行胰酶酶切，使特征性Ⅲ型胶原肽游离出来，然后通过液质联用仪检测是否含有该特征性Ⅲ型胶原肽，判定是否含有鸡源性成分。该方法特征性强，灵敏度高，操作简单，可对胶原水解物及其制品中的鸡源性成分进行测定。
Description
技术领域
本发明涉及鸡源的特征性Ⅲ型胶原肽及在胶原水解物和其制品检测中的应用，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、筋和腱等结缔组织中。明胶是胶原的水解物，是由动物的骨、皮、筋和腱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、牛和羊的皮或骨为原材料，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。
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特征肽，关系到溯源的成功与否。而对鸡源性成分溯源的特征肽的选择，不但要克服基因突变带来的影响，而且还需在鸡中含有，而其他动物中不含有。选择鸡中不突变的特征肽，成为鸡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，提供了鸡源的特征性Ⅲ型胶原肽及在胶原水解物和其制品中鸡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鸡源性成分的溯源鉴定和含量测定。
本发明的技术方案如下：
鸡源性成分的特征肽及在胶原水解物和其制品检测中的应用，基于鸡的Ⅲ型胶原蛋白序列中包含特征肽，其氨基酸序列为Gly-Ala-Gln-Gly-Pro-Pro-Gly-Pro-Thr-Gly-Ala-Arg或/和Gly-Ser-Hyp-Gly-Pro-Hyp-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Asp-Arg，其中Hyp为4羟基脯氨酸或3羟基脯氨酸。它是从牛、猪、羊、鸭与鸡中COL3A1蛋白的mRNA出发，找出鸡的稳定存在的特征mRNA序列，翻译成蛋白序列后利用液质联用技术检测，最终选定的特征肽。
该特征肽在胶原水解物及其制品检测中的应用，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；待测样品为胶原水解物或其制品，胶原水解物包括明胶等；
(2)放入液质联用仪，并以所述的稳定的特征肽或含有所述的稳定的特征肽的胰蛋白酶酶切后的鸡明胶作为对照品，选择对应的母离子m/z533.4和/或m/z669.6以及其子离子进行监测；如果检出该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，则所述待测样品胶原水解物或其制品含有鸡源性成分，鸡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，则不含有鸡源性成分。
上述明胶是由动物的皮、骨或鳞为原料经水解所制得的明胶，明胶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鸡源性成分。尽管动物中存在基因突变导致蛋白序列差异，但本发明的特征肽的基因在鸡中保守性强，且为鸡特有，通过该特征肽，可准确对胶原水解物及其制品中鸡源性成分进行溯源检测。
可通过检测其中一条或两条特征肽，来确定是否含有鸡源性成分。具体地，通过对样品中的蛋白成分进行胰酶酶切，使特征肽游离出来，然后通过液质联用仪检测是否含有该特征肽，来判定是否含有鸡源性成分。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鸡源性成分进行测定。
附图说明
图1是鸡明胶中特征肽Gly-Ala-Gln-Gly-Pro-Pro-Gly-Pro-Thr-Gly-Ala-Arg的子离子扫描质谱图(母离子m/z533.4，子离子扫描范围m/z200～1200)；
图2是各种动物明胶SRM扫描模式下选择离子对m/z533.4→809.6，752.7监测的质谱图；
图3是鸡、鸭、牛、猪、羊的COL3A1蛋白的部分mRNA序列及对应的比较。
图4是鸡明胶中特征肽Gly-Ser-Hyp-Gly-Pro-Hyp-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Asp-Arg的子离子扫描质谱图(母离子m/z669.6，子离子扫描范围m/z200～1400)；
图5是各种动物明胶SRM扫描模式下选择离子对m/z669.6→597.2，660.4监测的质谱图；
图6是以不同含量鸡明胶对应的特征肽Gly-Ala-Gln-Gly-Pro-Pro-Gly-Pro-Thr-Gly-Ala-Arg的质谱检测峰面积(m/z533.4→809.6)的关系曲线图。
图7是以不同含量鸡明胶对应的特征肽Gly-Ser-Hyp-Gly-Pro-Hyp-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Asp-Arg的质谱检测峰面积(m/z669.6→597.2)的关系曲线图。
图8是鸡明胶及明胶制品(以QQ糖为例)在SRM扫描模式下选择离子对m/z533.4→809.6，752.7监测的质谱图。
图9是鸡、鸭、牛、猪、羊的COL3A1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，分别来源于鸡、鸭、牛、猪、羊，分别从鸡皮、鸭皮、牛皮、猪皮、羊皮提取获得。
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特征肽的筛选与确定
经对鸡皮检测，发现鸡皮中含有大量的COL3A1蛋白，并且该蛋白具有一定的保守性，因此可以从COL3A1蛋白中筛选出一段肽作为鸡源性成分的特征肽。为了从基因中筛选出用于检测的稳定的特征肽，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，则相应地在鸭、牛、猪或羊中第一个碱基应为G或C；若第二个碱基为A或T，则相应地在鸭、牛、猪或羊中第二个碱基应为T或A；(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鸡的特征肽；如果有翻译后修饰则翻译后修饰的多肽即为鸡的特征肽；(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)特征肽的初筛
将鸡、鸭、牛、猪、羊的COL3A1蛋白对应的mRNA序列进行比对，部分序列展示如图3(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)。其中线框中鸡的mRNA序列“GGT GCA CAA GGT CCT CCA GGA CCT ACT GGA GCA AGA”中满足上述条件(a)、(b)、(c)。线框中鸡的mRNA序列，翻译后，对应的多肽为“Gly-Ala-Gln-Gly-Pro-Pro-Gly-Pro-Thr-Gly-Ala-Arg”，为确定该多肽Gly-Ala-Gln-Gly-Pro-Pro-Gly-Pro-Thr-Gly-Ala-Arg是否为鸡的特征肽，需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的鸡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+)，选择m/z533.4为母离子进行子离子全扫描。结果：以m/z533.4为母离子的子离子全扫描的质谱图与多肽Gly-Ala-Gln-Gly-Pro-Pro-Gly-Pro-Thr-Gly-Ala-Arg相符，见图1。结果表明鸡的mRNA序列“GGT GCA CAA GGT CCT CCA GGA CCT ACT GGAGCA AGA”翻译的肽Gly-Ala-Gln-Gly-Pro-Pro-Gly-Pro-Thr-Gly-Ala-Arg中没有脯氨酸发生羟基化。
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/z533.4→809.6，752.7作为检测离子对。结果见图2，6.6min处只有鸡明胶中检出相应的离子峰，其他均没有检出。
综上，多肽Gly-Ala-Gln-Gly-Pro-Pro-Gly-Pro-Thr-Gly-Ala-Arg可作为鸡源性成分的特征肽。
实施例2
与实施例1相同的方法。
1材料和试剂
材料：不同动物来源的明胶，分别来源于鸡、鸭、牛、猪、羊，分别从鸡皮、鸭皮、牛皮、猪皮、羊皮提取获得。
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检测方法
(1)特征肽的初筛
将鸡、鸭、牛、猪、羊的COL3A1蛋白对应的mRNA序列进行比对，部分序列展示如图9(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)。其中线框中鸡的mRNA序列“GGC AGC CCA GGT CCC CCT GGC CCA AGT GGA CCT GCGGGA GAC CGT”中满足实施例1中的条件(a)、(b)、(c)。线框中鸡的mRNA序列，翻译后，对应的多肽为“Gly-Ser-Pro-Gly-Pro-Pro-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Asp-Arg”，为确定该多肽Gly-Ser-Pro-Gly-Pro-Pro-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Asp-Arg是否为鸡的特征肽，需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的鸡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/z653.6、661.6和m/z669.6为母离子进行子离子全扫描。结果：(1)以m/z653.6为母离子的子离子全扫描的质谱图与目的多肽Gly-Ser-Pro-Gly-Pro-Pro-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Asp-Arg不符；(2)以m/z661.6为母离子的子离子全扫描的质谱图与目的多肽发生一个脯氨酸羟基化的也不符；(3)以m/z669.6为母离子的子离子全扫描的质谱图与目的多肽发生2个脯氨酸羟基化的一致，为Gly-Ser-Hyp-Gly-Pro-Hyp-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Asp-Arg(见图4)。结果表明鸡的mRNA序列“GGCAGC CCA GGT CCC CCT GGC CCA AGT GGA CCT GCG GGA GAC CGT”翻译的肽中有2个氨基酸被修饰了，即该多肽中有2个脯氨酸发生了羟基化，羟基化后的多肽序列为Gly-Ser-Hyp-Gly-Pro-Hyp-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Asp-Arg。
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/z669.6→597.2，660.4作为检测离子对。结果见图5，7.0min处只有鸡明胶中检出相应的离子峰，其他均没有检出。
综上，多肽Gly-Ser-Hyp-Gly-Pro-Hyp-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Asp-Arg可作为鸡源性成分的特征肽。
实施例3
1材料和试剂
材料：混合明胶样品(包括：含5％鸡明胶的猪明胶、10％鸡明胶的猪明胶、20％鸡明胶的猪明胶、40％鸡明胶的猪明胶，纯鸡明胶)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/z533.4→809.6，752.7作为检测离子对。
以鸡明胶的浓度为横坐标(X)，以峰面积(m/z533.4→809.6)为纵坐标(Y)，进行曲线绘制。结果见图6，标准曲线Y＝150428X+5544，线性相关系数R2＝0.9930，线性良好。可见该法可用于鸡源性成分的含量检测。
实施例4
1材料和试剂
材料：混合明胶样品(包括：含5％鸡明胶的猪明胶、10％鸡明胶的猪明胶、20％鸡明胶的猪明胶、40％鸡明胶的猪明胶、60％鸡明胶的猪明胶，纯鸡明胶)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/z669.6→597.2，660.4作为检测离子对。
以鸡明胶的浓度为横坐标(X)，以峰面积(m/z669.6→597.2)为纵坐标(Y)，进行曲线绘制。结果见图7，标准曲线Y＝152383X-11102，线性相关系数R2＝0.9966，线性良好。可见该法可用于鸡源性成分的含量检测。
实施例5
1材料和试剂
材料：鸡明胶、加入牛明胶的QQ糖(自制)、加入鸡明胶的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的鸡明胶、加入牛明胶的QQ糖、加入鸡明胶的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/z533.4→809.6，752.7作为检测离子对。
结果见图8，6.6min处鸡明胶和加入鸡明胶的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鸡源性成分。
Claims (5)
1.鸡源的特征性Ⅲ型胶原肽，其特征在于，其氨基酸序列为Gly-Ala-Gln-Gly-Pro-Pro-Gly-Pro-Thr-Gly-Ala-Arg或/和Gly-Ser-Hyp-Gly-Pro-Hyp-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Asp-Arg，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所述的鸡源的特征性Ⅲ型胶原肽，其特征在于，是从牛、猪、羊、鸭与鸡中COL3A1蛋白的mRNA出发，找出鸡的稳定存在的特征mRNA序列，翻译成蛋白序列后利用液质联用技术检测，最终选定的特征性Ⅲ型胶原肽。
3.权利要求1所述的鸡源的特征性Ⅲ型胶原肽的应用，其特征在于，在鸡的胶原降解物和其制品检测中的应用，进行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，具体方法包括以下步骤：
(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；待测样品为胶原降解物或其制品，胶原降解物包括明胶；
(2)放入液质联用仪，并以所述特征性Ⅲ型胶原肽或含有所述特征性Ⅲ型胶原肽的胰蛋白酶酶切后的鸡明胶作为对照品，选择对应的母离子m/z533.4和/或m/z669.6以及其子离子进行监测；如果检出该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，则所述待测样品胶原降解物或其制品含有鸡源性成分，鸡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，则不含有鸡源性成分。
5.按照权利要求4所述的应用，其特征在于，明胶是由动物的皮、骨或鳞为原料经水解所制得的明胶，明胶制品是原料中含有明胶的食品、保健品或药品。
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