色谱 ›› 2021, Vol. 39 ›› Issue (5): 463-471.DOI: 10.3724/SP.J.1123.2020.08019

• 研究论文 • 上一篇    下一篇

样品制备方法对高效液相色谱-串联质谱分析人乳内源肽的影响

于文皓1, 于洋1, 王雯丹2, 李依彤2, 司徒文佑2, 靳艳1,*()   

  1. 1.中国科学院大连化学物理研究所, 中国科学院分离分析重点实验室, 辽宁 大连 116023
    2.北京伊利科技发展有限责任公司, 北京 100070
  • 收稿日期:2020-10-13 出版日期:2021-05-08 发布日期:2021-03-31
  • 通讯作者: 靳艳
  • 作者简介:Tel:(0411)84379910,E-mail:yanjin@dicp.ac.cn.
  • 基金资助:
    大连化物所创新研究基金项目(DICP I201906);内蒙古自治区重大基础研究开放课题

Effect of sample preparation on analysis of human milk endogenous peptides using liquid chromatography-tandem mass spectrometry

YU Wenhao1, YU Yang1, WANG Wendan2, LI Yitong2, SZETO Ignatius M.2, JIN Yan1,*()   

  1. 1. CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical and Physics, Chinese Academy of Sciences, Dalian 116023, China
    2. Beijing Yili Technology Development Co., Ltd., Beijing 100070, China
  • Received:2020-10-13 Online:2021-05-08 Published:2021-03-31
  • Contact: JIN Yan
  • Supported by:
    Innovation Research Fund of Dalian Institute of Chemical and Physics(DICP I201906);Inner Mongolia Autonomous Region Basic Research Project

摘要:

人乳内源肽是乳蛋白在乳腺中被降解形成的具有生理功能的肽,是人乳的重要组成部分,研究人乳内源肽对于婴儿健康具有重要的意义。高效液相色谱-串联质谱(LC-MS/MS)联用技术的应用,促使人乳内源肽的研究取得了突破性的进展。人乳中内源肽含量低、干扰组分多,样品制备方法是影响分析结果的关键步骤。为了研究样品制备方法对分析结果的影响,分别采用不变性超滤法(UF 1)、热变性超滤法(UF 2)、化学变性超滤法(UF 3)、三氯乙酸沉淀法(PCPN 1)、乙醇沉淀法(PCPN 2)、强疏水性碳介孔材料(highly ordered mesoporous carbon, OMC)富集法等6种方法从人乳中提取内源肽,利用LC-MS/MS研究样品制备方法对人乳内源肽分析结果的影响。结果表明,UF 1和UF 2法制备的样品中可鉴定到的肽段数目分别为1161±8条和1017±91条,两种方法制备的样品中肽序列的重合率大于70%, UF 1在所有方法中鉴定到的肽的数目最多。UF 3法制备的样品所能鉴定到的肽段数目最少,仅为366±18条。PCPN 1和PCPN 2两种沉淀法制备样品中的内源肽分别为779±69和876±55条,但内源肽差异较大,仅有约50%肽段序列重合。OMC法制备样品中肽的数目为549±151条,与其他方法相比,虽然鉴定的肽数量上没有优势,但该方法制备的样品中肽在等电点(pI)和亲水性平均系数(GRAVY)等性质上没有偏倚,说明该法可用于制备特定人乳内源肽。6种方法制备的样品中鉴定到来源于β-酪蛋白、免疫球蛋白受体、骨桥蛋白、αS1-酪蛋白、κ-酪蛋白和胆盐激活脂肪酶的肽,并且源于以上蛋白质的肽段总数在该样品中均超过88%,说明6种方法制备的样品都可以满足鉴定一般人乳内源肽的需求。UF 2、UF 3和OMC法制备的样品中鉴定到源于乳铁蛋白的内源肽的数目分别为21、38和19条,内源肽在乳铁蛋白上的覆盖率分别为14%、16%和19%,而文献常用的PCPN 1法制备的样品则会丢失此类内源肽。综上,UF 2法制备的样品不仅肽段数量多、母体蛋白质种类丰富,还可鉴定到源于乳铁蛋白的肽,可作为人乳内源肽组学研究中的首选方法。

关键词: 液相色谱, 串联质谱, 内源肽, 人乳, 提取方法

Abstract:

Hundreds of endogenous peptides were released from milk proteins within the human mammary gland and some of them possess a variety of bioactive functions. Thus, it is important to investigate human milk endogenous peptides for infant health. Peptidomics based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been used to investigate human milk endogenous peptides. Extraction of endogenous peptides from human milk is an essential and key procedure for analyzing human milk peptides using LC-MS/MS. This study aimed to compare methods for extracting endogenous peptides from human milk using LC-MS/MS. Ultrafiltration methods including that not involving denaturation (UF 1), that involving heat denaturation (UF 2), and that involving chemical denaturation (UF 3), precipitation methods using trichloroacetic acid (PCPN 1) and alcohol (PCPN 2), and an enrichment method using highly ordered mesoporous carbon (OMC) were used to extract endogenous peptides from human milk. Extracted endogenous peptides were then analyzed using LC-MS/MS. The samples extracted using UF 1 and UF 2 comprised 1161±8 and 1017±91 endogenous peptides, respectively. More than 70% peptide sequences in each sample extracted using UF 1 and UF 2 overlapped. The results revealed that endogenous peptides extracted using UF 1 and UF 2 showed similar characteristics. UF 1 yielded the highest number of peptides, whereas UF 3 extracted the least number of peptides at 366±18. The number of endogenous peptides extracted using PCPN 1 and PCPN 2 were 779±69 and 876±55, respectively. However, their characteristics were quite different, and only about 50% peptide sequences overlapped. The number of peptides extracted using OMC (549±151) was not remarkable compared with that using other methods. However, the isoelectric point (pI) and grand average of hydropathicity (GRAVY) of the peptides extracted using OMC were different from those extracted using other methods. This method presented no selectivity for the endogenous peptides with different pI and GRAVY and may be used to extract unique peptides from human milk. A total of 205 peptides were commonly identified in the samples using each of the six methods. The percent of shared peptides across the six samples ranged from 13% to 23%. The number of unique peptides in the samples extracted using UF 1 and UF 2 (226 and 228, respectively) were the highest among those extracted using the six methods. The results showed that all six methods could be used to extract endogenous peptides from these high-abundance precursor proteins. A total of 21, 38, and 19 peptides were extracted from lactotransferrin using UF 2, UF 3, and OMC, respectively, and the coverage rates of these peptides in lactotransferrin were 14%, 16%, and 19%, respectively. These three methods could extract the endogenous peptides from lactotransferrin in human milk, but PCPN 1 that has been commonly used in previous studies could not. The peptides from β-casein, polymeric immunoglobulin receptor, osteopontia, αS1-casein, κ-casein, and bile salt-activated lipase were identified in all samples extracted using the six methods. Moreover, these precursor proteins contributed 88% peptides in the samples extracted using the six methods. In conclusion, UF 1 and UF 2 were efficient procedures for extracting endogenous peptides from human milk. In addition, UF 2 could extract peptides from lactotransferrin, which is the optimum choice for extracting endogenous peptides from human milk. Additionally, the OMC enrichment method can be used to enrich and extract specific endogenous peptides from human milk. This study systematically compared the sample preparation methods commonly used in human milk endogenous peptidomics in recent years. The results provide strong support for uniform and standardized sample preparation methods. An ultrafiltration method without denaturation, which is more advantageous than the currently commonly used trichloroacetic acid precipitation method, was also established to prepare human milk endogenous peptide samples. In combination with OMC, this method can help in a more comprehensive and in-depth understanding of the endogenous peptidome of human milk.

Key words: liquid chromatography (LC), tandem mass spectrometry (MS/MS), endogenous peptides, human milk, extraction method

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