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

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

孙浩峰^{1, 2,},
马凌云¹,
刘健仪¹,
张琦¹,
宋德伟^1, ,

1.
中国计量科学研究院，北京 100029
2.
南京理工大学，南京 210094

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

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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

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

SUN Haofeng^{1, 2
,},
MA Lingyun¹,
LIU Jianyi¹,
ZHANG Qi¹,
SONG Dewei^{1
, ,}

1.
National Institute of Metrology, Beijing 100029, China
2.
Nanjing University of Science and Technology, Nanjing 210000, China

摘要

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。
- 计量学 /
- 心肌肌钙蛋白I /
- 抗原表位 /
- 氢氘交换 /
- 质谱 /
- 体外诊断试剂
Abstract: Acute myocardial infarction (AMI) ranks first among the death factors in China, and is the top priority in the clinical prevention and treatment of cardiovascular diseases. Myocardial troponin I (cTnI), as the most reliable clinical index of evaluating myocardial injury, is currently the preferred biomarker and gold standard for the diagnosis of myocardial infarction. Immunology method is a common method of clinical detection AMI, many domestic and foreign diagnostic reagent suppliers can produce cTnI detection kit, but immune testing system from different manufacturers will produce different value results for the same sample, on the one hand, the commercial kits of different suppliers in sensitivity, specificity and selectivity, on the other hand is different kits used in different antibodies, identify antigen epitope region, antibody and serum cTnI sample binding ability differences, resulting in inconsistent results between different manufacturers, thus affect the accuracy of clinical diagnosis. The consistency and accuracy of the quantitative results are closely related to the structure of cTnI. However, due to the complexity of cTnI, the structural characterization of cTnI in the solution state is very challenging. Through experimental condition optimization and data processing parameters, the characterization method of cTnI HDX, based on hydrogen deuterium exchange-mass spectrometry (HDX-MS) technology, is established, and applied to the study of cTnI. Meanwhile, this method has good universality and can be easily extended to cTnI conformation transition study and the development of kinetic method, which lays the technical foundation for promoting the standardization of cTnI from the perspective of protein structure.
- metrology /
- cardiac troponin I /
- epitope /
- H/D exchange /
- mass spectrometry /
- in vitro diagnosis

HTML全文

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

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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

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图 3 cTnI的肽图

Figure 3. Peptide map of cTnI

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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

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图 5 表位区域的氘吸收速率曲线

Figure 5. Deuterium absorption rate curve for epitope region

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图 6 保护性区域在肌钙蛋白上的映射

Figure 6. Mapping of protective regions on troponin

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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)	肽段产量
0	0	0	0
1.6	0	0.1	0
3.2	0	0.1	0
0	4	0.1	66

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表 2 PLGS参数

Table 2. PLGS parameter

项目	处理参数
Acquisition Type	Electrospray MS^e
Lock Mass for Charge 1	556.2771
Low Energy Threshold	130
Search Type	Electrospray MS^e
Peptide Tolerance	100
Fragment Tolerance	100
Primary Digest Reagent	Non-specific

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