CN112213401B - 一种阿胶及其制品的驴源性特征肽及其阿胶及其制品的鉴别方法 - Google Patents

一种阿胶及其制品的驴源性特征肽及其阿胶及其制品的鉴别方法 Download PDF

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CN112213401B
CN112213401B CN201910620749.9A CN201910620749A CN112213401B CN 112213401 B CN112213401 B CN 112213401B CN 201910620749 A CN201910620749 A CN 201910620749A CN 112213401 B CN112213401 B CN 112213401B
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刘睿
郑云枫
段金廒
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Nanjing University of Chinese Medicine
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Abstract

本发明公开了一种用于鉴别阿胶及其制品的驴源性特征肽，所述的肽段包括肽段一:HGNRGEPGPVGSVGPVGAVGPRGPSGPQGVRGDK和/或肽段二:GPTGEPGK。本发明通过大量实验筛选得到两条驴源性特征肽段，这两条特征肽段具有很高的专属性，可用于鉴定驴皮、阿胶及阿胶制品。并且本发明提供的阿胶及其制品的鉴别方法，通过大量实验筛选出最佳的色谱条件和质谱条件，整个方法操作简单，准确性高，有利于阿胶的质量控制，可大大提高阿胶及其产品的检测效率和准确度，对保证阿胶及其制品的质量具有重要的意义。

Description

一种阿胶及其制品的驴源性特征肽及其阿胶及其制品的鉴别 方法
技术领域
本发明属于医药与食品检测技术领域，具体涉及阿胶及其制品的驴源性特征肽及其阿胶及其制品的鉴别方法。
背景技术
阿胶(Asini Corii Colla)为马科动物驴Equus asinus L.的干燥皮或鲜皮经煎煮、浓缩制成的固体胶。阿胶具补血滋阴，润燥，止血之功。用于血虚萎黄，眩晕心悸，肌痿无力，心烦不眠，虚风内动，肺燥咳嗽，劳嗽咯血，吐血尿血，便血崩漏，妊娠胎漏。《本草纲目》载：“阿胶《本经》上品。弘景曰：‘出东阿，故名阿胶’”。道地阿胶表面平整，无油气孔，质硬而脆，断面光亮细腻，碎片对光照视呈棕色半透明状，李时珍赞其“黄透如琥珀色，光黑如瑿漆”。
在现有市场上存在假阿胶、伪劣阿胶，并以次充好向市场销售，严重影响了广大人民群众的饮食和用药安全。
目前不法企业常以马皮、牛皮、猪皮等掺杂制备阿胶来以次充好，导致阿胶造假事件屡禁不止的原因之一是缺少简单、可行、准确的鉴定技术支持，动物皮经过熬制后，很难鉴别出动物皮的来源。因此，寻找专属性、准确高的方法来鉴定阿胶及其制品真伪意义重大。
发明内容
发明目的：本发明基于阿胶与其它多种伪品胶的多肽成分对比分析，从中筛选辨识两个阿胶特征肽段，并建立以液相色谱-串连质谱技术进行阿胶真伪鉴别的方法。本发明可填补阿胶及其制品真伪鉴别的空白，为保证阿胶及其制品的质量提供科学方法。
技术方案：为了实现以上目的，本发明采取的技术方案为：
一种用于鉴别阿胶及其制品的驴源性特征肽，其所述的特征肽序列为：
肽段一:
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pr o-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys和/或肽段二:Gly-Pro-Thr-Gly-Glu-Pro-Gly-Lys。
作为优选方案，以上所述的用于鉴别阿胶及其制品的驴源性特征肽，其中脯氨酸Pro可被羟基化形成羟脯氨酸Hyp。
作为优选方案，以上所述的用于鉴别阿胶及其制品的特征肽，其中天门冬酰胺Asn及谷氨酰胺Gln可被脱酰胺化分别形成天冬氨酸Asp及谷氨酸Glu。
一种阿胶及其制品的鉴别方法，其包括以下步骤：
(1)以缓冲液或水溶解待测的阿胶及阿胶制品，加入1％～5％的蛋白内切酶LysC在37℃酶解12-18小时，获得多肽溶液，多肽溶液脱盐后浓缩干燥；
(2)采用LC-MS/MS检测，流动相A相为3％乙腈水溶液，其中含0.1％三氟乙酸，流动相B相为80％乙腈水溶液，其中含0.1％三氟乙酸，梯度洗脱，100min，0％流动相B→30％流动相B；
(3)质谱扫描范围：100-2000Da，正离子模式，选择多电荷进行二级质谱分析；
(4)对获得的MS/MS图谱，采用搜库软件进行分析，自建蛋白质数据库，自建蛋白质数据库中包含除了以上所述的两条驴源性特征肽的信息外，还包含猪、牛、马、鹿动物的同源肽段信息；
(5)搜库参数中PTM的设置包括：羟基化，脱酰胺化和氧化
(6)根据搜库结果作出判断：
搜库结果中
a.样品搜库结果有且仅有驴源来源的肽段His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys和/或Gly-Pro-Thr-Gly-Glu-Pro-Gly-Lys的信息，则为驴源性正品；
b、样品搜库结果未见驴源来源的肽段His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys和/或Gly-Pro-Thr-Gly-Glu-Pro-Gly-Lys的信息，则为伪品；
c、样品搜库结果有驴源来源的肽段His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys和/或Gly-Pro-Thr-Gly-Glu-Pro-Gly-Lys的信息，且有其它动物猪、牛、马、鹿的同源肽段信息，则为掺杂样品。
一种阿胶及其制品的鉴别方法，其包括以下步骤：
(1)阿胶对照溶液的制备：取阿胶对照药材，加pH值为7～8的缓冲溶液溶解，经蛋白内切酶LysC酶解，然后经Seppak C18脱盐处理，制备得到阿胶对照药材溶液；
(2)待检测样品溶液的制备：取待测阿胶或阿胶制品，按照步骤(1)相同的方法，制备得到待检测样品溶液；
(3)将步骤(1)和(2)得到的鹿胶对照溶液及待测样品溶液，采用液相色谱-串连质谱法，以电喷雾正离子模式扫描，对带电荷大于2的肽段进行二级质谱扫描，获得MS/MS图谱；
(4)将获得的MS/MS图谱，采用搜库软件(Peaks 8.5软件)进行分析，自建蛋白质数据库，自建数据库中包含除了两条驴源性特征肽段的信息外，还可包含猪、牛、马、鹿等动物的同源肽段信息；
(5)自建的蛋白质数据库如下：
>Deer_Peptide_1
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Asp-Lys
>Cattle_Peptide_1
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Asp-Lys
>Donkey_Peptide_1
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys
>Pig_Peptide_1
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ser-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Glu-Lys
>Sheep_Peptide_1
His-Gly-Ser-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Asp-Lys
>Horse_Peptide_1
His-Gly-His-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys
>Deer_Peptide_2
Gly-Pro-Thr-Gly-Asp-Pro-Gly-Lys
>Cattle_Peptide_2
Gly-Pro-Ser-Gly-Asp-Pro-Gly-Lys
>Donkey_Peptide_2
Gly-Pro-Thr-Gly-Glu-Pro-Gly-Lys
>Pig_Peptide_2
Gly-Pro-Thr-Gly-Asp-Pro-Gly-Lys
>Sheep_Peptide_2
Gly-Pro-Thr-Gly-Asp-Pro-Gly-Lys
>Horse_Peptide_2
Gly-Pro-Ser-Gly-Glu-Pro-Gly-Lys
(6)搜库参数中PTM的设置包括：羟基化，脱酰胺化和氧化；
(7)根据搜库结果作出判断：
搜库结果中
a.如果有且仅有驴源来源的肽段His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys和/或Gly-Pro-Thr-Gly-Glu-Pro-Gly-Lys，则为阿胶制品；
b.如果除了驴源来源的肽段His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys和/或Gly-Pro-Thr-Gly-Glu-Pro-Gly-Lys外，还含有其它任何肽段，则为掺假阿胶样品；
c.如果未检测到驴源来源的肽段His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys和/或Gly-Pro-Thr-Gly-Glu-Pro-Gly-Lys，则为阿胶伪品。
作为优选方案，以上所述的阿胶及其制品的前处理方法为LysC蛋白酶进行酶切，由于LysC选择性的切断赖氨酸(Lys，K)后面的酰胺键，与胰蛋白酶选择性切断精氨酸(Arg，R)与赖氨酸(Lys，K)后面的酰胺键不同，本发明通过实验发现，采用LysC蛋白酶进行酶切，才可获得更具有专属性的肽段，可用于鉴别与区分阿胶及其制品。
作为优选方案，以上所述的阿胶及其制品的鉴别方法，步骤(3)的色谱条件为：
戴安U3000 NanoRSLC纳升液相系统，色谱柱为5μm Reprosil C18AQ，规格为75μm×150mm，上样量为2μg，流速300nL/min，流动相A为体积比为2：0.2：98的乙腈/甲酸/水，流动相B为体积比为80/0.2/20的乙腈/甲酸/水，0～150min，2～30％B线性梯度洗脱；
质谱条件为：喷雾电压为2.5kV，离子传输毛细管温度为200℃；质谱一级全扫描范围为m/z 300～2000，分离宽度为3Da；串联质谱分析采用一级质谱数据依赖的二级质谱扫描模式，依次选取一级质谱中离子强度最高的5个离子进行碰撞诱导解离二级串联质谱。
本发明所述的阿胶及其制品的鉴别方法，所述的阿胶制品包括：阿胶膏、阿胶糕、阿胶口服液、阿胶浆、阿胶颗粒。
本发明所述的阿胶及其制品的鉴别方法，阿胶伪品包括驴皮、马皮、猪皮、牛皮、鹿皮、羊皮等。
有益效果：本发明通过大量实验筛选，筛选出两条驴源性特征肽段，这两条特征肽段具有很高的专属性，可用于鉴定驴皮、阿胶及阿胶制品。
本发明提供的阿胶及其制品的鉴别方法，通过大量实验筛选出最佳的色谱条件和质谱条件。整个方法操作简单，判断准确，能准确区分阿胶与马皮胶。本发明提供的肽段序列及技术方法有利于阿胶的质量控制，且可鉴别市场上的各类阿胶制品，可大大提高阿胶及其产品的检测效率和准确度，对保证阿胶及其制品的质量具有重要的意义。
附图说明
图1为本发明提供的用于鉴别阿胶及其制品的驴源性特征肽的质谱图。
具体实施方式
下面结合具体实施例进一步阐明本发明，应理解这些实施例仅用于说明本发明而不用于限制本发明的范围，在阅读了本发明之后，本领域技术人员对本发明的各种等价形式的修改均落于本申请所附权利要求所限定的范围。
实施例1：阿胶、马皮胶、黄明胶(牛皮胶)、新阿胶(猪皮胶)、鹿皮胶、羊皮胶的鉴别
(1)收集阿胶、马皮胶、黄明胶(牛皮胶)、新阿胶(猪皮胶)、鹿皮胶和羊皮胶，取各种胶类药材粉末0.1g，置于50ml锥形瓶中加入1％ NH4HCO3溶液定容至刻度，使其溶解，配制LysC蛋白酶溶液(LysC酶100μg用Resuspension Buffer 1ml重新溶解)，取胶类药液200μl加入配制好的LysC蛋白酶溶液20μl，37℃酶解12h，加入10％三氟乙酸溶液10μl进行灭活，离心(10000r/min)，用Seppak C18固相萃取脱盐，样品液氮气吹干，加水溶解，备用。
(2)然后采用液相色谱-串连质谱法，以电喷雾正离子模式扫描，对带电荷大于2的肽段进行二级质谱扫描，获得MS/MS图谱；
Nano LC-MS/MS条件：
戴安U3000 NanoRSLC纳升液相系统，色谱柱为5μm Reprosil C18AQ(75μm×150mm)，上样量为2μg，流速300nL/min，流动相A(乙腈/甲酸/水＝2/0.2/98，v/v/v)，流动相B(乙腈/甲酸/水＝80/0.2/20，v/v/v)，2～30％B线性梯度洗脱150min。
质谱条件为：喷雾电压为2.5kV，离子传输毛细管温度为200℃；质谱一级全扫描范围为m/z 300～2000，分离宽度为3Da；串联质谱分析采用一级质谱数据依赖的二级质谱扫描模式，依次选取一级质谱中离子强度最高的5个离子进行碰撞诱导解离(CID)二级串联质谱。
选择自建数据库搜索，数据库中包含：
>Deer_Peptide_1
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Asp-Lys
>Cattle_Peptide_1
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Asp-Lys
>Donkey_Peptide_1
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys
>Pig_Peptide_1
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ser-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Glu-Lys
>Sheep_Peptide_1
His-Gly-Ser-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Asp-Lys
>Horse_Peptide_1
His-Gly-His-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys
>Deer_Peptide_2
Gly-Pro-Thr-Gly-Asp-Pro-Gly-Lys
>Cattle_Peptide_2
Gly-Pro-Ser-Gly-Asp-Pro-Gly-Lys
>Donkey_Peptide_2
Gly-Pro-Thr-Gly-Glu-Pro-Gly-Lys
>Pig_Peptide_2
Gly-Pro-Thr-Gly-Asp-Pro-Gly-Lys
>Sheep_Peptide_2
Gly-Pro-Thr-Gly-Asp-Pro-Gly-Lys
>Horse_Peptide_2
Gly-Pro-Ser-Gly-Glu-Pro-Gly-Lys
检索参数设置为：前体离子误差20ppm；子离子误差0.2Da；允许2个位点误切，假阳性率(FDR)≤1％；酶切方式选择胰酶(LysC)，唯一肽段数(unique peptides)≥2；其它参数为默认参数，在上述检索条件下所得分值有显著性意义(P<0.05)被认定为有效的鉴定结果。
结果阿胶样品中仅鉴定出专属肽段His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys(质谱图如图1所示)与Gly-Pro-Thr-Gly-Glu-Pro-Gly-Lys，马皮胶中仅鉴定出肽段His-Gly-His-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys与Gly-Pro-Ser-Gly-Glu-Pro-Gly-Lys。
鹿皮胶中鉴定出His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Asp-Lys与Gly-Pro-Thr-Gly-Asp-Pro-Gly-Lys，牛皮胶中鉴定出His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Asp-Lys与Gly-Pro-Ser-Gly-Asp-Pro-Gly-Lys，羊皮胶中鉴定出His-Gly-Ser-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Asp-Lys与Gly-Pro-Thr-Gly-Asp-Pro-Gly-Lys，猪皮胶中鉴定出His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ser-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Glu-Lys与Gly-Pro-Thr-Gly-Asp-Pro-Gly-Lys，通过本发明的方法可准确鉴定出阿胶。
实施例2：马皮掺假阿胶的鉴别
(1)将马皮胶以不同比例掺入到阿胶中(1：99、20：80、50：50)，取掺假阿胶样品粉末0.1g，置于50ml锥形瓶中加入1％ NH4HCO3溶液定容至刻度，使其溶解，配制LysC蛋白酶溶液(LysC酶100μg用Resuspension Buffer 1ml重新溶解)，取胶类药液200μl加入配制好的LysC蛋白酶溶液20μl，37℃酶解12h，加入10％三氟乙酸溶液10μl进行灭活，离心(10000r/min)，用Seppak C18固相萃取脱盐，样品液氮气吹干，加水溶解，备用。
(2)然后采用液相色谱-串连质谱法，以电喷雾正离子模式扫描，对带电荷大于2的肽段进行二级质谱扫描，获得MS/MS图谱；
Nano LC-MS/MS条件：
戴安U3000 NanoRSLC纳升液相系统，色谱柱为5μm Reprosil C18AQ(75μm×150mm)，上样量为2μg，流速300nL/min，流动相A(乙腈/甲酸/水＝2/0.2/98，v/v/v)，流动相B(乙腈/甲酸/水＝80/0.2/20，v/v/v)，2～30％B线性梯度洗脱150min。
质谱条件为：喷雾电压为2.5kV，离子传输毛细管温度为200℃；质谱一级全扫描范围为m/z 300～2000，分离宽度为3Da；串联质谱分析采用一级质谱数据依赖的二级质谱扫描模式，依次选取一级质谱中离子强度最高的5个离子进行碰撞诱导解离(CID)二级串联质谱。
选择自建数据库搜索，数据库中包含：
>Donkey_Peptide_1
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys
>Horse_Peptide_1
His-Gly-His-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys
>Donkey_Peptide_2
Gly-Pro-Thr-Gly-Glu-Pro-Gly-Lys
>Horse_Peptide_2
Gly-Pro-Ser-Gly-Glu-Pro-Gly-Lys
检索参数设置为：前体离子误差20ppm；子离子误差0.2Da；允许2个位点误切，假阳性率(FDR)≤1％；酶切方式选择胰酶(LysC)，唯一肽段数(unique peptides)≥2；其它参数为默认参数，在上述检索条件下所得分值有显著性意义(P<0.05)被认定为有效的鉴定结果。
掺假的阿胶样品中除了检测到阿胶的专属肽段外，均检测到马皮的专属性肽段His-Gly-His-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys与Gly-Pro-Ser-Gly-Glu-Pro-Gly-Lys，表明本发明方法可检验有马皮掺假的阿胶样品，掺假量为1％即可检出。
实施例3：以骡子皮为原料制备的阿胶伪品(骡皮胶)的鉴别
(1)取骡皮胶样品粉末0.1g，置于50ml锥形瓶中加入1％ NH4HCO3溶液定容至刻度，使其溶解，配制LysC蛋白酶溶液(LysC酶100μg用Resuspension Buffer 1ml重新溶解)，取胶类药液200μl加入配制好的LysC蛋白酶溶液20μl，37℃酶解12h，加入10％三氟乙酸溶液10μl进行灭活，离心(10000r/min)，用Seppak C18固相萃取脱盐，样品液氮气吹干，加水溶解，备用。
(2)然后采用液相色谱-串连质谱法，以电喷雾正离子模式扫描，对带电荷大于2的肽段进行二级质谱扫描，获得MS/MS图谱；
Nano LC-MS/MS条件：
戴安U3000 NanoRSLC纳升液相系统，色谱柱为5μm Reprosil C18AQ(75μm×150mm)，上样量为2μg，流速300nL/min，流动相A(乙腈/甲酸/水＝2/0.2/98，v/v/v)，流动相B(乙腈/甲酸/水＝80/0.2/20，v/v/v)，2～30％B线性梯度洗脱150min。
质谱条件为：喷雾电压为2.5kV，离子传输毛细管温度为200℃；质谱一级全扫描范围为m/z 300～2000，分离宽度为3Da；串联质谱分析采用一级质谱数据依赖的二级质谱扫描模式，依次选取一级质谱中离子强度最高的5个离子进行碰撞诱导解离(CID)二级串联质谱。
选择自建数据库搜索，数据库中包含：
>Donkey_Peptide_1
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys
>Horse_Peptide_1
His-Gly-His-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys
>Donkey_Peptide_2
Gly-Pro-Thr-Gly-Glu-Pro-Gly-Lys
>Horse_Peptide_2
Gly-Pro-Ser-Gly-Glu-Pro-Gly-Lys
检索参数设置为：前体离子误差20ppm；子离子误差0.2Da；允许2个位点误切，假阳性率(FDR)≤1％；酶切方式选择胰酶(LysC)，唯一肽段数(unique peptides)≥2；其它参数为默认参数，在上述检索条件下所得分值有显著性意义(P<0.05)被认定为有效的鉴定结果。
从骡子皮制备的阿胶伪品中同时检测到了马皮与驴皮的专属性肽段His-Gly-His-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys与His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys，表明以骡皮制备阿胶伪品，或以骡皮掺假阿胶制品，其中可检测出马皮的专属性肽段，即可认为被测样品为非阿胶及其制品。
实施例4：多种动物皮掺假阿胶的鉴别
(1)将马皮胶、牛皮胶、鹿皮胶、猪皮胶与阿胶等比例掺到一起，取掺假阿胶样品粉末0.1g，置于50ml锥形瓶中加入1％ NH4HCO3溶液定容至刻度，使其溶解，配制LysC蛋白酶溶液(LysC酶100μg用Resuspension Buffer 1ml重新溶解)，取胶类药液200μl加入配制好的LysC蛋白酶溶液20μl，37℃酶解12h，加入10％三氟乙酸溶液10μl进行灭活，离心(10000r/min)，用C18固相萃取脱盐，样品液氮气吹干，加水溶解，备用。
(2)然后采用液相色谱-串连质谱法，以电喷雾正离子模式扫描，对带电荷大于2的肽段进行二级质谱扫描，获得MS/MS图谱；
Nano LC-MS/MS条件：
戴安U3000 NanoRSLC纳升液相系统，色谱柱为5μm Reprosil C18AQ(75μm×150mm)，上样量为2μg，流速300nL/min，流动相A(乙腈/甲酸/水＝2/0.2/98，v/v/v)，流动相B(乙腈/甲酸/水＝80/0.2/20，v/v/v)，2～30％B线性梯度洗脱150min。
质谱条件为：喷雾电压为2.5kV，离子传输毛细管温度为200℃；质谱一级全扫描范围为m/z 300～2000，分离宽度为3Da；串联质谱分析采用一级质谱数据依赖的二级质谱扫描模式，依次选取一级质谱中离子强度最高的5个离子进行碰撞诱导解离(CID)二级串联质谱。
选择自建数据库搜索，数据库中包含：
>Deer_Peptide_1
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Asp-Lys
>Cattle_Peptide_1
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Asp-Lys
>Donkey_Peptide_1
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys
>Pig_Peptide_1
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ser-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ar g-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Glu-Lys
>Horse_Peptide_1
His-Gly-His-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys
>Deer_Peptide_2
Gly-Pro-Thr-Gly-Asp-Pro-Gly-Lys
>Cattle_Peptide_2
Gly-Pro-Ser-Gly-Asp-Pro-Gly-Lys
>Donkey_Peptide_2
Gly-Pro-Thr-Gly-Glu-Pro-Gly-Lys
>Pig_Peptide_2
Gly-Pro-Thr-Gly-Asp-Pro-Gly-Lys
>Horse_Peptide_2
Gly-Pro-Ser-Gly-Glu-Pro-Gly-Lys
检索参数设置为：前体离子误差20ppm；子离子误差0.2Da；允许2个位点误切，假阳性率(FDR)≤1％；酶切方式选择胰酶(LysC)，唯一肽段数(unique peptides)≥2；其他参数为默认参数，在上述检索条件下所得分值有显著性意义(P<0.05)被认定为有效的鉴定结果。
掺假的阿胶样品中除了检测到阿胶的专属肽段(His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys与Gly-Pro-Thr-Gly-Glu-Pro-Gly-Lys)外，还检测到马皮胶的专属肽段His-Gly-His-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys与Gly-Pro-Ser-Gly-Glu-Pro-Gly-Lys，以及其它动物皮的肽段。表明本发明的鉴别方法，不仅可用于检测马皮掺杂阿胶，还可检测多种动物皮掺假的阿胶样品。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 南京中医药大学
<120> 一种阿胶及其制品的驴源性特征肽及其阿胶及其制品的鉴别方法
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 34
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
His Gly Asn Arg Gly Glu Pro Gly Pro Val Gly Ser Val Gly Pro Val
1 5 10 15
Gly Ala Val Gly Pro Arg Gly Pro Ser Gly Pro Gln Gly Val Arg Gly
20 25 30
Asp Lys
<210> 2
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Gly Pro Thr Gly Glu Pro Gly Lys
1 5

Claims (1)

1.一种阿胶及其制品的鉴别方法，其特征在于，包括以下步骤：
（1）收集阿胶、马皮胶、牛皮胶、猪皮胶、鹿皮胶和羊皮胶，取各种胶类药材粉末0.1g，置于50ml锥形瓶中加入1% NH4HCO3溶液定容至刻度，使其溶解，将LysC酶100μg用Resuspension Buffer 1ml重新溶解，配制成LysC蛋白酶溶液；取以上各胶类药液200μl加入配制好的LysC蛋白酶溶液20μl，37℃酶解12h，加入10%三氟乙酸溶液10μl进行灭活，10000 r/min离心，用Seppak C18固相萃取脱盐，样品液氮气吹干，加水溶解，备用；
（2）然后采用液相色谱-串连质谱法，以电喷雾正离子模式扫描，对带电荷大于2的肽段进行二级质谱扫描，获得MS/MS图谱；
Nano LC-MS/MS条件：
戴安U3000 NanoRSLC纳升液相系统，色谱柱为5 μm Reprosil C18AQ，规格75μm ×150 mm，上样量为2μg，流速300 nL/min，流动相A相为体积比2：0.2：98的乙腈/甲酸/水，流动相B为体积比80/0.2/20的乙腈/甲酸/水，2~30%B线性梯度洗脱150 min；
质谱条件为：喷雾电压为2.5 kV，离子传输毛细管温度为200℃；质谱一级全扫描范围为m/z 300~2000，分离宽度为3 Da；串联质谱分析采用一级质谱数据依赖的二级质谱扫描模式，依次选取一级质谱中离子强度最高的5个离子进行碰撞诱导解离二级串联质谱；
选择自建数据库搜索，数据库中包含：
>Deer_Peptide_1
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Asp-Lys
>Cattle_Peptide_1
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Asp-Lys
>Donkey_Peptide_1
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys
>Pig_Peptide_1
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ser-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Glu-Lys
>Sheep_Peptide_1
His-Gly-Ser-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Asp-Lys
>Horse_Peptide_1
His-Gly-His-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys
>Deer_Peptide_2
Gly-Pro-Thr-Gly-Asp-Pro-Gly-Lys
>Cattle_Peptide_2
Gly-Pro-Ser-Gly-Asp-Pro-Gly-Lys
>Donkey_Peptide_2
Gly-Pro-Thr-Gly-Glu-Pro-Gly-Lys
>Pig_Peptide_2
Gly-Pro-Thr-Gly-Asp-Pro-Gly-Lys
>Sheep_Peptide_2
Gly-Pro-Thr-Gly-Asp-Pro-Gly-Lys
>Horse_Peptide_2
Gly-Pro-Ser-Gly-Glu-Pro-Gly-Lys
检索参数设置为：前体离子误差20 ppm；子离子误差0.2 Da；允许2个位点误切，假阳性率≤1%；酶切方式选择胰酶LysC，唯一肽段数≥2；
阿胶样品中仅鉴定出专属肽段：
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys与Gly-Pro-Thr-Gly-Glu-Pro-Gly-Lys；
马皮胶中仅鉴定出肽段：
His-Gly-His-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ser-Val-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Val-Arg-Gly-Asp-Lys与Gly-Pro-Ser-Gly-Glu-Pro-Gly-Lys；
鹿皮胶中鉴定出肽段：
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Asp-Lys与Gly-Pro-Thr-Gly-Asp-Pro-Gly-Lys；
牛皮胶中鉴定出肽段：
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Asp-Lys与Gly-Pro-Ser-Gly-Asp-Pro-Gly-Lys；
羊皮胶中鉴定出肽段：
His-Gly-Ser-Arg-Gly-Glu-Pro-Gly-Pro-Val-Gly-Ala-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Asp-Lys与Gly-Pro-Thr-Gly-Asp-Pro-Gly-Lys；
猪皮胶中鉴定出肽段：
His-Gly-Asn-Arg-Gly-Glu-Pro-Gly-Pro-Ala-Gly-Ser-Val-Gly-Pro-Ala-Gly-Ala-Val-Gly-Pro-Arg-Gly-Pro-Ser-Gly-Pro-Gln-Gly-Ile-Arg-Gly-Glu-Lys与Gly-Pro-Thr-Gly-Asp-Pro-Gly-Lys。
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