CN111735966B - 一种液相色谱串联质谱法检测意蜂蜜和中蜂蜜中王浆主蛋白5的方法 - Google Patents
一种液相色谱串联质谱法检测意蜂蜜和中蜂蜜中王浆主蛋白5的方法 Download PDFInfo
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- CN111735966B CN111735966B CN202010715105.0A CN202010715105A CN111735966B CN 111735966 B CN111735966 B CN 111735966B CN 202010715105 A CN202010715105 A CN 202010715105A CN 111735966 B CN111735966 B CN 111735966B
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Abstract
本发明提供一种液相色谱串联质谱法检测中蜂蜜和意蜂蜜的王浆主蛋白5(MRJP5)的方法，包括：筛选分别属于中蜂MRJP5和意蜂MRJP5的特征肽段，建立液相色谱串联质谱检测方法，蜂蜜样品前处理，液相色谱分离以及串联质谱检测，数据分析等步骤；所述特征肽段包括意蜂MRJP5特征肽段：YLDYDFGSDER和中蜂MRJP5特征肽段：NLANSMNVIHEWK。本发明所提出的蜂蜜中MRJP5的检测方法，具有较高的准确度、精确度和灵敏度，并且稳定性强，适用于鉴别意蜂蜜和中蜂蜜，尤其适用于中蜂蜜混入意蜂蜜的掺假鉴别。该方法对保护蜂蜜消费者的权益和维护蜂产品行业的健康发展具有重要现实意义。
Description
技术领域
本发明涉及食品检测领域，具体为一种液相色谱串联质谱法检测蜂蜜中王浆主蛋白5的方法，该方法适用于鉴别中蜂蜜和意蜂蜜。
背景技术
蜂蜜是由蜜蜂采集蜜源植物的花蜜，与自身分泌物相混合、经充分酿造贮存在蜂巢中的一种天然甜味物质。蜂蜜中含有丰富的氨基酸、酚酸、黄酮、微量元素等对人有益的物质，这使其具有润肠、美容和保健等功效，深受人们喜爱。根据蜜源植物的不同，蜂蜜可以分为洋槐蜜、椴树蜜、枣花蜜、荆条蜜、荔枝蜜、龙眼蜜等单花蜜品种。根据蜜蜂种类的不同，可以分为西方蜜蜂、东方蜜蜂、大蜜蜂、小蜜蜂、黑小蜜蜂、黑大蜜蜂、无刺蜂等蜜蜂酿制的蜂蜜。当前，已经人工驯化的蜜蜂，并且实现大规模生产蜜蜂的蜜蜂品种主要是以意大利蜜蜂为代表的西方蜜蜂和以中华蜜蜂为代表的东方蜜蜂，其它蜜蜂品种尚未人工驯化，难以开展规模化养殖。
目前，中蜂蜜由于口感佳，深受人们喜爱，但是中蜂蜜产量较低，仅仅是我国商品蜂蜜的有效补充，也因此其售价往往是意蜂蜜的5-10倍。不良企业为了追去高额利润，往往制售掺假中蜂蜜，其掺假的形式主要是在中蜂蜜中混入廉价的意蜂蜜。当前，尚未有任何有关中蜂蜜真实性的标准或掺假鉴别方法，如何评价中蜂蜜的真实性，鉴别中蜂蜜掺假，已经成为蜂产品行业广大从业者共识。
蜜蜂在酿制蜂蜜的过程中会混入少量的酶类、蛋白质等物质，由于蜜蜂种类的不同，酿制的蜂蜜中含有的蛋白质也会有所不同。王浆主蛋白(MRJPs)是蜂蜜中蛋白质的主要组成成分，中蜂和意蜂酿制的蜂蜜虽然均含有MRJPs，但是两者的氨基酸序列存在差异。通过检测中蜂蜜和意蜂蜜中的同源性蛋白质可以有效的鉴别蜜蜂的品种，从而对酿制的蜜蜂进行品种溯源，进而用来中蜂蜜真实性的评价和惨假鉴别方法的开发。本发明针对蜂蜜丰度较高的MRJP5，基于液相串联质谱开发了中蜂和意蜂MRJP5的检测方法。建立的方法尤其适用于中蜂蜜中意蜂蜜掺假的鉴别，这对保护中蜂蜜产业的发展具有重大的现实意义。
发明内容
为了解决现有技术存在的问题，本发明提供了一种液相色谱串联质谱法检测蜂蜜中王浆主蛋白5的方法。
该方法可用于生产和商业中对中蜂蜜和意蜂蜜进行鉴定。
第一方面，本发明筛选出可用于液相色谱串联质谱鉴定中蜂蜜和意蜂蜜的王浆主蛋白5（MRJP5）的特征肽段组：
意蜂蜜MRJP5的特征肽段：YLDYDFGSDER，
中蜂蜜MRJP5的特征肽段：NLANSMNVIHEWK。
本发明根据意蜂蜜和中蜂蜜酶解产物的纳升级高效液相色谱液相色谱串联质谱检测结果，成功筛选出意蜂蜜特征肽段：YLDYDFGSDER，中蜂蜜特征肽段：NLANSMNVIHEWK，两者特异性通过Uniprot数据库进行了验证。
进一步，意蜂蜜的MRJP5的特征肽段YLDYDFGSDER在质谱中所产生的检测信号的母离子具有690.29114的质荷比，包含质荷比为1103.42761、710.31039的子离子。
中蜂蜜的MRJP5的特征肽段NLANSMNVIHEWK在质谱中所产生的检测信号的母离子具有519.26104的质荷比，包含质荷比为712.37769、599.29362的子离子。
第二方面，本发明提供一种液相色谱串联质谱检测蜂蜜中MRJP5的方法。
具体地，所述方法具体步骤如下：
A、根据筛选出的专属于意蜂蜜的MRJP5的特征肽段和中蜂蜜的MRJP5的特征肽段的母离子质荷比、子离子质荷比、碰撞能等信息建立液相色谱串联质谱检测方法；
B、提取待测蜂蜜中的蛋白质，用胰蛋白酶进行酶切，对酶解产物除盐后作为待测样本，使用液相色谱串联质谱检测；
C、在质谱检测结果中提取筛选的特征肽段，以鉴别蜂蜜为意蜂蜜或中蜂蜜。
进一步地，步骤A和B中采用UHPLC-Q Exactive plus进行液相色谱串联质谱检测。
a1、液相色谱条件如下：
色谱柱：为C18柱；
流动相组成：流动相A为0.1%甲酸水溶液，流动相B为含0.1%甲酸的乙腈；
梯度洗脱条件为：0-0.5 min，5%的B；0.5-1.0 min，5-15%的B；1.0-6.5 min，15-40%的B；6.5-7.0 min，40-95%的B；7.0-8.5 min，95%的B；8.5-8.6 min，95-5%B，8.6-10.0min，5%的B；流速：0.30 mL/min；进样量：5.0 µL；
a2、质谱条件如下：
离子源：纳升电喷雾离子源；一级扫描方式：正离子扫描；扫描分辨率：120000；自动增益控制（AGC Target）：3e6；扫描范围：300~1800 m/z；动态隔离窗口：20s。二级扫描参数：裂解模式：高能碰撞诱导解离模式（HCD）；扫描分辨率：17500；扫描范围：200~2000 m/z；Loop count：20；MSX count：1；最大离子注入时间：35 ms；自动增益控制（AGC Target）：1e5；隔离窗口：2.0 m/z。
该方法采用UHPLC-Q Exactive Plus（超高效液相色谱-四级杆串联高分辨静电轨道阱）检测蜂蜜样本，通过在待测样品的质谱图中提取属于中蜂MRJP5特征肽段和意蜂MRJP5特征肽段的质荷比，若待测样品在正确的保留时间中仅提取出中蜂MRJP5特征肽段，且子离子也符合，则可判断为中蜂蜜；若待测样品在正确的保留时间中仅提取出意蜂MRJP5特征肽段，且子离子也符合，则可判断为意蜂蜜；若待测样品在正确的保留时间中同时提取出中蜂和意蜂MRJP5特征肽段，且子离子也符合，则判断为中、意蜂蜜混掺。
应当理解的是，对上述所用试剂或原料的用量进行等比例扩大或者缩小后的技术方案，与上述内容实质等同，均属于本发明保护范围。
本发明采用UHPLC-Q Exactive plus仪器检测蜂蜜中的MRJP5，基于高分辨质谱提供的精确质量数，该方法具有较高的特异性和灵敏度。基于本方法采用的仪器和参数，不同分析实验室和检测机构可以依据液相串联高分辨质谱技术的相关知识，对其中的参数进行一定调整，上述调整均属于本发明保护范围。
本发明提供一种液相色谱串联质谱检测中蜂蜜和意蜂蜜MRJP5的方法，至少具有如下有益效果：
本发明经研究发现，意蜂MRJP5和中蜂MRJP5的氨基酸位点有区别，可以确定各自的特异性的肽段，并据此建立起一套鉴别中蜂蜜和意蜂蜜的方法，用于辅助蜂蜜掺假的鉴别。本发明提供的中、意蜂蜜中MRJP5的检测方法，具有特异性强、灵敏度高、准确度和精确度好等优点。该方法对于维护蜂蜜消费行业健康发展和蜂蜜消费者的权益有重要意义。
附图说明
图1为本发明实施例1提供的通过UHPLC-Q Exactive plus提取的意蜂MRJP5特征肽段和中蜂MRJP5的特征肽段的离子流图；
图2为本发明实施例1提供的通过UHPLC-Q Exactive plus检测的意蜂MRJP5特征肽段和中蜂MRJP5的特征肽段的质谱图；
图3为本发明实施例1提供的通过UHPLC-Q Exactive plus检测的意蜂MRJP5特征肽段和中蜂MRJP5的特征肽段的二级碎片质谱图；
图4为本发明实施例1提供的MRJP5的各特征肽段响应对比图；
图5为本发明实施例2提供的通过UHPLC-Q Exactive plus检测的意蜂蜜掺假检测结果图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
以下实施例中涉及的仪器与试剂：
1.超高静电场轨道阱傅里叶变换台式质谱高分辨质谱仪（LTQ-Orbitrap Elite），美国Thermo Fisher Scientific公司；
2. 纳升级高效液相色谱系统（Easy-nLC 1000），美国Thermo Fisher Scientific公司；
3.台式低温离心机(Microfuge 22R Centrifuge)，美国 BeckMAN Coul TER公司；
4.全波长酶标仪(Multiskan GO)，美国Thermo Fisher Scientific公司；
5.电子分析天平((PL203)，德国METTLERTOLEDO公司；
6. pH计(DELTA 320)，德国METTLER TOLEDO公司；
7.蒸发浓缩仪(Speed-Vacsvstem, RVC2-18)，德国 MarinChrist公司；
8.超纯水机(Milli-QGradient)，美国Millipore公司；
9.超低温冰箱(MDF-U3286S)，日本SANYO公司；
10.1290 Infinity 液相色谱-6495三重四级杆质谱，美国Agilent Technologies公司；
11.二硫苏糖醇(DL-Dithiothreitol, DTT)，中国Solarbio公司；
12.碘乙酰胺(Iodoacetamide, IAA)，美国Merk公司；
13.Bradford法蛋白质定量试剂盒，中国Solarbio公司；
14.碳酸氢铵(NH4HCO3)，美国Sigma公司。
实施例1
1.样品来源
从市场或者蜂农处购买真实的蜂蜜样本共20份。
2.实验步骤
2.1溶液配制
5M Urea溶液：称取Urea 4.5 g，超纯水定容到15 mL；
100 mM DTT：称取DTT 308.5 mg，40 mM NH4HCO3溶液定容到20 mL，每管l mL分装，-20℃冰箱保存待用。
40 mM NH4HCO3溶液：称取0.316 g NH4HCO3，用超纯水定容100 mL，4℃冰箱保存待用。
100 mM IAA溶液：称取IAA 0.39 g，用40 mM的NH4HCO3溶液定溶至20 mL，-20℃冰箱保存待用。
活化液：500 μL ACN、3 μL TFA，超纯水定容至1 mL，4℃保存。
平衡液:1 μL TFA，超纯水定容至1 mL，存于4℃。
洗脱液：800 μL ACN、1 μL TFA，用超纯水定容至1 mL，存于4℃。
2.2本发明对蜂蜜酶切后得到的肽段进行液相色谱串联质谱检测（纳升级高效液相色谱系统串联超高静电场轨道阱傅里叶变换台式质谱高分辨质谱仪），然后将质谱结果导入PEAK软件中进行肽段的匹配，从结果中筛选得到意蜂MRJP5的特征肽段：YLDYDFGSDER；中蜂蜜MRJP5的特征肽段：NLANSMNVIHEWK。
图1-图3分别为特征肽段的离子流图、质谱图和二级碎片质谱图。
对蜂农处采集的真实蜂蜜样本的MRJP5的肽段进行匹配检测，结果整理为图4，从图4中可以看出在蜂蜜中，本发明筛选意蜂蜜MRJP5的特征肽段：YLDYDFGSDER的响应最高，由此可以体现其含量相对较高；同理筛选出中蜂蜜MRJP5的特征肽段： NLANSMNVIHEWK的响应最高，由此可以体现其含量相对较高，可以作为检测蜂蜜样本中MRJP5的肽段。
2.3蜂蜜样本的前处理
（1）准确称取均匀待测蜂蜜10 g至50 mL离心管中，加入10 mL 去离子水，涡旋至蜂蜜充分溶解。于12000 rpm，4℃条件下离心10 min。收集上清液于新的2 mL离心管中。
（2）移取蛋白质溶液100 μL与400 μL 40 mM NH4HCO3混合。向上述混合溶液中加入50 μL 30 mM DTT溶液，在室温反应60 min，然后再加入250 μL 100 mM IAA溶液在室温下暗反应60 min。
（3）每个样品中加入30 μL胰蛋白酶溶液，于37℃过夜酶切。当酶切反应完成时，加入1 μL FA以使胰蛋白酶失活。然后将酶切产物进行除盐，除盐后的样品进入质谱分析。
（4）蜂蜜样本的质谱分析
使用1290 Infinity 液相色谱-6495三重四级杆质谱对蜂蜜样本进行检测，质谱液相条件如下：
色谱柱：Kinetex C18 (50 x 2.1 mm) 2.6µm，柱温为室温：20 ℃。
流动相组成：流动相A为0.1%的甲酸水，流动相B为0.1%的甲酸乙腈。梯度洗脱条件为：0-0.8 min，10%的B；0.8-1.3 min，10-20%的B；1.3-4.5 min，20-30%的B；4.5-5.4 min，30-95%的B；5.4-6.0min，95%的B；6.0-6.1 min，95-5%B，6.1-7.0 min，5%的B。
流速：0.30 mL/min；进样量：5.0 µL。
所述的质谱条件如下：
离子源参数：鞘气温度350 ℃；鞘气流速12 L/min；辅助气温度290 ℃；辅助气流速11 L/min；毛细管电压正模式3.5kV；iFunnel参数正模式高压200V、低压100V，采集的模式为正离子模式下的MRM模式。
（5）蜂蜜样本的数据处理
如图1-图3所示，检测蜂蜜样本的图谱中应该含有意蜂MRJP5的特征肽段：YLDYDFGSDER，其精确m/z值： 690.29114（[M+2H]2+）；或者包含中蜂蜜MRJP5的特征肽段：NLANSMNVIHEWK，其精确m/z值：519.26104（[M+2H]2+），其允许偏差应在10 ppm以内。
其子离子图谱中应该含有意蜂蜜MRJP5的特征肽段：YLDYDFGSDER，其特征碎片离子m/z 1103.42761、710.31039；或者包含中蜂蜜MRJP5的特征肽段：NLANSMNVIHEWK，其特征碎片离子m/z 712.37769、599.29362。且其精确质量数的误差应当小于5 ppm。蜂蜜样本中只有在精确m/z值和特征碎片离子方面同时满足以上的特征，方可肯定该蜂蜜样本中的MRJP5检测结果可信，以此鉴别中蜂蜜和意蜂蜜。
实施例2 蜂蜜掺假的检测
1、样品来源
从市场或者蜂农处购买真实的中蜂蜜、意蜂蜜样本。
2、实验步骤
（1）溶液配制
同实施例1。
（2）蜂蜜的混掺：按不同比例向中蜂蜜中掺入意蜂蜜。
向蜂蜜中按照5%、10%、20%、30%、40%、50%的比例掺入意蜂蜜。
（3）蜂蜜样本的前处理
①准确称取均匀待测蜂蜜10 g至50 mL离心管中，加入10 mL 去离子水，涡旋至蜂蜜充分溶解。于12000 rpm，4℃条件下离心10 min。收集上清液于新的2 mL离心管中。
②移取蛋白质溶液200 μL与800 μL 40 mM NH4HCO3混合。向上述混合溶液中加入100 μL 30 mM DTT溶液，在室温反应60 min，然后再加入500 μL 100 mM IAA溶液在室温下暗反应60 min。
③每个样品中加入30 μL胰蛋白酶溶液，于37℃过夜酶切。当酶切反应完成时，加入1 μL FA以使胰蛋白酶失活。然后将酶切产物进行除盐，除盐后的样品进入质谱分析。
（4）蜂蜜样本的质谱分析
使用超高效液相串联高分辨质谱（UHPLC-Q Exactive plus）对蜂蜜样本进行检测。
（5）蜂蜜样本的数据处理
从质谱数据中提取特征肽段的质荷比，以m/z 690.29114的峰面积作为对比依据。结果如图5所示，结果表明本发明能够检测出掺入意蜂蜜为5%以上的蜂蜜掺假现象。
实施例3 商品中蜂蜜的检测
1、样品来源
从商场购买的不同厂家的中蜂蜜30瓶。
2、实验步骤
（1）溶液配制
同实施例1。
（2）待测样本的前处理
①准确称取均匀待测蜂蜜10 g至50 mL离心管中，加入10 mL 去离子水，涡旋至蜂蜜充分溶解。于12000 rpm，4℃条件下离心10 min。收集上清液于新的离心管中。
②移取蛋白质溶液200 μL与800 μL 40 mM NH4HCO3混合。向上述混合溶液中加入100 μL 30 mM DTT溶液，在室温反应60 min，然后再加入500 μL 100 mM IAA溶液在室温下暗反应60 min。
③每个样品中加入30 μL胰蛋白酶溶液，于37℃过夜酶切。当酶切反应完成时，加入1 μL FA以使胰蛋白酶失活。然后将酶切产物进行除盐，除盐后的样品进入质谱分析。
（4）蜂蜜样本的质谱分析
使用超高效液相串联高分辨质谱（UHPLC-Q Exactive plus）对蜂蜜样本进行检测。
（5）蜂蜜样本的数据处理
从质谱数据中提取特征肽段的质荷比，以m/z 690.29114和519.26104的峰面积作为对比依据。结果如表1所示，结果表明目前蜂蜜市场的中蜂蜜掺假现象严重。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
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Claims (5)
1.一种液相色谱串联质谱法检测中蜂蜜和意蜂蜜的王浆主蛋白5的方法，其特征在于，包括：在对待测蜂蜜进行预处理后，通过液相色谱串联质谱法检测待测蜂蜜中王浆主蛋白5的特征肽段，所述特征肽段包括肽段:意蜂蜜特征肽段：YLDYDFGSDER；中蜂蜜特征肽段：NLANSMNVIHEWK；
后在质谱图中提取属于所述中蜂蜜特征肽段和所述意蜂蜜特征肽段的质荷比，若待测蜂蜜在正确的保留时间中仅提取出所述中蜂蜜特征肽段，且子离子也符合，则可判断为中蜂蜜；若待测蜂蜜在正确的保留时间中仅提取出所述意蜂蜜特征肽段，且子离子也符合，则可判断为意蜂蜜；若待测蜂蜜在正确的保留时间中同时提取出了所述中蜂蜜特征肽段和所述意蜂蜜特征肽段，且子离子也符合，则判断为中、意蜂蜜混掺；
所述预处理包括如下流程：将待测蜂蜜以10g:10mL的比例溶解于去离子水中，离心取上清得到蛋白质溶液；
将所述蛋白质溶液以100 μL: 400 μL的比例与40 mM NH4HCO3混合，并加入50μL的30mM DTT溶液，室温反应60min，然后再加入DTT溶液5倍体积的100 mM IAA溶液在室温下暗反应60 min；
再加入胰蛋白酶溶液，37℃过夜酶切，所述胰蛋白酶溶液和所述蛋白质溶液比例为30μL: 100 μL。
2.根据权利要求1所述的方法，其特征在于，所述方法具体包括以下步骤：
A、对真实的中蜂蜜和意蜂蜜进行酶解，酶解产物除盐后干燥，用0.1%的甲酸水溶液复溶，然后进行液相色谱串联质谱检测；
B、根据液相色谱串联质谱检测结果，筛选出专属于意蜂蜜的王浆主蛋白5的特征肽段和中蜂蜜的王浆主蛋白5的特征肽段；
C、提取待测蜂蜜中的蛋白质，用胰蛋白酶进行酶切，对酶解产物除盐后作为待测样本，使用液相色谱串联质谱检测；
D、在质谱检测结果中提取筛选的特征肽段，以鉴别蜂蜜为意蜂蜜或中蜂蜜。
3.根据权利要求2所述的方法，其特征在于，步骤A中采用纳升级高效液相色谱系统串联超高静电场轨道阱傅里叶变换台式质谱高分辨质谱仪；步骤C中采用三重四级杆或QExactive plus液质联用仪进行液相色谱串联质谱检测。
4.根据权利要求3所述的方法，其特征在于，意蜂蜜的王浆主蛋白5的特征肽段YLDYDFGSDER在质谱中所产生的检测信号的母离子具有m/z 690.29114的质荷比，包含质荷比为m/z 1103.42761、m/z 710.31039的子离子，检测允许偏差在10 ppm以内；中蜂蜜的王浆主蛋白5的特征肽段NLANSMNVIHEWK在质谱中所产生的检测信号的母离子具有m/z 519.26104的质荷比，包含质荷比为m/z 712.37769、m/z 599.29362的子离子。
5.权利要求1-4任一项所述方法在意蜂蜜和中蜂蜜真实性评价中的应用。
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| CN101434954A (zh) * | 2008-12-22 | 2009-05-20 | 浙江大学 | 中华蜜蜂王浆主蛋白AccMRJP7基因及其编码蛋白 |
| CN106879884A (zh) * | 2015-12-15 | 2017-06-23 | 中国农业大学 | 一种提高蜂王浆性能的方法 |
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