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肌钙蛋白I的氢氘交换表征方法研究

孙浩峰 马凌云 刘健仪 张琦 宋德伟

孙浩峰,马凌云,刘健仪,等. 肌钙蛋白I的氢氘交换表征方法研究[J]. 计量科学与技术,待出版 doi: 10.12338/j.issn.2096-9015.2024.0166
引用本文: 孙浩峰,马凌云,刘健仪,等. 肌钙蛋白I的氢氘交换表征方法研究[J]. 计量科学与技术,待出版 doi: 10.12338/j.issn.2096-9015.2024.0166
SUN Haofeng, MA Lingyun, LIU Jianyi, ZHANG Qi, SONG Dewei. Research on Characterization Method of Cardiac Troponin I by Hydrogen-Deuterium Exchange Mass Spectrometry[J]. Metrology Science and Technology. doi: 10.12338/j.issn.2096-9015.2024.0166
Citation: SUN Haofeng, MA Lingyun, LIU Jianyi, ZHANG Qi, SONG Dewei. Research on Characterization Method of Cardiac Troponin I by Hydrogen-Deuterium Exchange Mass Spectrometry[J]. Metrology Science and Technology. doi: 10.12338/j.issn.2096-9015.2024.0166

肌钙蛋白I的氢氘交换表征方法研究

doi: 10.12338/j.issn.2096-9015.2024.0166
基金项目: 国家重点研发计划(2022YFF0710301)。
详细信息
    作者简介:

    孙浩峰(1995-),南京理工大学博士研究生,研究方向:检验医学溯源及标准化,邮箱:Haofeng_Sun@163.com

    通讯作者:

    宋德伟(1976-),中国计量科学研究院研究员,研究方向:检验医学溯源及标准化,邮箱:songdw@nim.ac.cn

Research on Characterization Method of Cardiac Troponin I by Hydrogen-Deuterium Exchange Mass Spectrometry

  • 摘要: 急性心肌梗死(AMI)位于我国人群死亡因素排名之首,是临床上心血管疾病防治的重中之重。心肌肌钙蛋白I(cTnI)作为评价心肌损伤最可靠的临床指标,是目前诊断心梗的首选生物标志物和金标准。免疫学方法是临床检测AMI的常用方法,国内外众多诊断试剂供应商可以生产cTnI检测试剂盒,然而来自不同制造商的免疫测试系统对同一样本会产生不同的定值结果,一方面原因是不同供应商的商用免疫试剂盒在敏感性、特异性和选择性方面不同,另一方面原因是不同试剂盒所使用抗体不同,识别抗原表位区域不同,抗体与血清cTnI样本结合能力有差异,导致不同厂商试剂盒间定值结果不一致,从而影响临床诊断的准确性。定量结果的一致性和准确性与cTnI的结构息息相关。然而受限于cTnI的复杂性,溶液状态下cTnI的结构表征十分具有挑战性。通过实验条件优化和数据处理参数,建立了基于氢氘交换-质谱(HDX-MS)技术的cTnI HDX表征方法,并将其应用cTnI的抗原表位研究,同时该方法具有较好的普适性,可扩展至cTnI构象转变研究及动力学方法的开发等,为从蛋白结构角度促进cTnI的标准化奠定了技术基础。
  • 图  1  进样量对肽段生成数量及序列覆盖率的影响

    Figure  1.  Effect of injection volume on peptide generation and sequence coverage

    图  2  cTnI在线酶解的总离子流图和基峰色谱图

    Figure  2.  TIC and BPI map of cTnI on-line digestion

    图  3  cTnI的肽图

    Figure  3.  Peptide map of cTnI

    图  4  游离cTnI和与15Y1和23S2结合后的氘吸收热图

    Figure  4.  Deuterium absorption heat map of free cTnI and complexes of 15Y1 and 23S2

    图  5  表位区域的氘吸收速率曲线

    Figure  5.  Deuterium absorption rate curve for epitope region

    图  6  保护性区域在肌钙蛋白上的映射

    Figure  6.  Mapping of protective regions on troponin

    表  1  猝灭缓冲液组成对在线酶切效率的影响

    Table  1.   Effect of quenching buffer composition on on-line digestion efficiency

    变性剂还原剂肽段产量
    GdnHCl
    (mol/L)
    Urea
    (mol/L)
    TCEP
    (mol/L)
    0000
    1.6 00.1 0
    3.2 00.1 0
    040.166
    下载: 导出CSV

    表  2  PLGS参数

    Table  2.   PLGS parameter

    项目 处理参数
    Acquisition Type Electrospray MSe
    Lock Mass for Charge 1 556.2771
    Low Energy Threshold 130
    Search Type Electrospray MSe
    Peptide Tolerance 100
    Fragment Tolerance 100
    Primary Digest Reagent Non-specific
    下载: 导出CSV
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出版历程
  • 收稿日期:  2024-05-22
  • 录用日期:  2024-06-03
  • 修回日期:  2024-06-03
  • 网络出版日期:  2024-07-16

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