超高效液相色谱-串联质谱法定量检测蜂王浆主蛋白1～3

doi:10.3785/j.issn.1008-9209.2022.07.272

浙江大学学报（农业与生命科学版）

2022, Vol. 48

Issue (6): 776-786 DOI: 10.3785/j.issn.1008-9209.2022.07.272

研究论文

超高效液相色谱-串联质谱法定量检测蜂王浆主蛋白1～3

陈勇^1,²(

),江唯健¹,王加俊²,张帆¹,沈立荣^1,²(

)

^1.浙江大学生物系统工程与食品科学学院，杭州 310058
^2.杭州蜂之语蜂业股份有限公司，杭州 311500

Quantitative detection of major royal jelly proteins 1-3 by ultra-high performance liquid chromatography-tandem mass spectrometry

Yong CHEN^1,²(

),Weijian JIANG¹,Jiajun WANG²,Fan ZHANG¹,Lirong SHEN^1,²(

)

^1.College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China
^2.Hangzhou Beewords Apiculture Co. , Ltd. , Hangzhou 311500, China

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摘要：

为建立精准的蜂王浆新鲜度检测方法，利用超高效液相色谱-串联四极杆飞行时间质谱（ultra-high performance liquid chromatography-tandem quadrupole-time-of-flight mass spectrometry, UPLC-Q-TOF/MS）对经胰蛋白酶酶解的蜂王浆中王浆主蛋白（major royal jelly proteins, MRJPs）进行定性分析，并通过蛋白质组学分析平台比对，鉴定出8种王浆主蛋白（MRJP1～7和MRJP9）的一级序列数据，然后按同时具备高易酶解性与酶解后高稳定性的标准筛选得到3个特异性标志多肽，包括MRJP1多肽YNGVPSSLNVISK、MRJP2多肽TLQMIAGMK、MRJP3多肽LTVAGESFTVK。分别合成MRJP1～3的3条特异性标志多肽标准品及其同位素标志肽，建立蜂王浆中MRJP1～3的超高效液相色谱-串联三重四极杆质谱（ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry, UPLC-TQMS）定量检测方法。采用UPLC-TQMS分别检测蜂王浆样品中MRJP1～3含量发现，MRJP1多肽在10～1 600 ng/mL范围内具有良好的线性关系，MRJP2和MRJP3多肽在10～600 ng/mL范围内均具有良好的线性关系，决定系数（R²）均大于0.99。最后，通过分析蜂王浆中MRJP1～3随加温老化时间的降解程度，证实MRJP1～3的降解率均与老化时间（40 ℃）呈正相关（R²＞0.9）。说明本研究所建立的MRJP1～3检测方法具有高度特异性，可为蜂王浆质量评价提供技术支撑。

关键词： 蛋白质组学; 超高效液相色谱-串联质谱法; 王浆主蛋白标志多肽; 蜂王浆; 新鲜度; 检测方法

Abstract:

In order to establish an accurate detection method for royal jelly freshness, the major royal jelly proteins (MRJPs) in royal jelly by trypsin enzymatic hydrolysis were identified by ultra-high performance liquid chromatography-tandem quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS). The primary sequence data of eight MRJPs (MRJP1-7 and MRJP9) were identified by proteomics analysis platform. Then three specific marker polypeptides were obtained according to the criteria of highly easy enzymolysis and high stability, including YNGVPSSLNVISK (MRJP1), TLQMIAGMK (MRJP2), and LTVAGESFTVK (MRJP3). The three specific marker polypeptides of MRJP1-3 and their isotopic marker peptides were synthesized, respectively, and an ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-TQMS) method for the quantitative detection of MRJP1-3 in royal jelly was established. The results showed that the MRJP1 polypeptide had a good linear relationship in the range of 10-1 600 ng/mL, and MRJP2 and MRJP3 polypeptides had a good linear relationship in the range of 10-600 ng/mL, and the correlations of determination (R²) were all more than 0.99. Finally, by analyzing the degradation degrees of MRJP1-3 in royal jelly with the aging time, it was confirmed that the degradation rates of MRJP1-3 were all positively correlated with the aging time at 40 ℃ (R²＞0.9). It is indicated that the MRJP1-3 detection method established in this study is highly specific, which provides technical support for the quality evaluation of royal jelly.

Key words: proteomics ultra-high performance liquid chromatography-tandem mass spectrometry marker polypeptide of major royal jelly proteins (MRJPs) royal jelly freshness detection method

收稿日期: 2022-07-27 出版日期: 2022-12-25

CLC:

S 896.3

基金资助: 浙江省科技计划项目(2017C32033);杭州市钱江特聘专家项目(202038)

通讯作者: 沈立荣 E-mail: 11413030@zju.edu.cn;shenlirong@zju.edu.cn

作者简介: 陈勇（https//orcid.org/0000-0001-8240-1286），E-mail：11413030@zju.edu.cn

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引用本文:

陈勇,江唯健,王加俊,张帆,沈立荣. 超高效液相色谱-串联质谱法定量检测蜂王浆主蛋白1～3[J]. 浙江大学学报（农业与生命科学版）, 2022, 48(6): 776-786.

Yong CHEN,Weijian JIANG,Jiajun WANG,Fan ZHANG,Lirong SHEN. Quantitative detection of major royal jelly proteins 1-3 by ultra-high performance liquid chromatography-tandem mass spectrometry. Journal of Zhejiang University (Agriculture and Life Sciences), 2022, 48(6): 776-786.

链接本文:

https://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2022.07.272 或 https://www.zjujournals.com/agr/CN/Y2022/V48/I6/776

图1 基于UPLC-TQMS的MRJPs候选多肽的稳定性分析

表1 MRJPs及其酶解多肽鉴定分析结果

图2 候选多肽的酶解性与酶解后稳定性热图#表示非特异性多肽，表2同。

表2 MRJPs候选多肽分类

表3 MRJP1～3的候选标志多肽

图3 MRJP1～3同位素标志肽的二级质谱图

图4 3条标志多肽的标准曲线

表4 鲜蜂王浆在40 ℃贮存过程中MRJP1～3含量变化 (mg/g)

图5 MRJP1、MRJP2和MRJP3降解率与老化时间（40 ℃）的线性关系

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