[发明专利]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

权利要求书

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。