超高效液相色谱-质谱联用法监测人心房钠尿肽的化学合成

doi:10.3724/SP.J.1123.2019.10020

色谱 ›› 2020, Vol. 38 ›› Issue (3): 324-331.DOI: 10.3724/SP.J.1123.2019.10020

超高效液相色谱-质谱联用法监测人心房钠尿肽的化学合成

徐坤¹^,², 金钰龙¹^,², 黄嫣嫣¹^,²^,^*(), 赵睿¹^,²^,^*()

¹ 北京分子科学国家研究中心, 中国科学院活体分析化学重点实验室, 中国科学院分子科学科教融合卓越创新中心, 中国科学院化学研究所, 北京 100190
² 中国科学院大学, 北京 100049

收稿日期:2019-10-20 出版日期:2020-03-08 发布日期:2020-12-10
通讯作者: 黄嫣嫣,赵睿
作者简介:赵睿, Tel:(010)62557910, E-mail:zhaorui@iccas.ac.cn
黄嫣嫣, Tel:(010)62557910, E-mail:yyhuang@iccas.ac.cn;
基金资助:
国家自然科学基金项目(21705154);国家自然科学基金项目(21675161);国家自然科学基金项目(21874141)

Monitoring chemical synthesis of human atrial natriuretic peptide by ultra high performance liquid chromatography-mass spectrometry analysis

XU Kun¹^,², JIN Yulong¹^,², HUANG Yanyan¹^,²^,^*(), ZHAO Rui¹^,²^,^*()

¹ Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Analytical Chemistry for Living Biosystems, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China
² University of Chinese Academy of Sciences, Beijing 100049, China

Received:2019-10-20 Online:2020-03-08 Published:2020-12-10
Contact: HUANG Yanyan,ZHAO Rui
Supported by:
National Natural Science Foundation of China(21705154);National Natural Science Foundation of China(21675161);National Natural Science Foundation of China(21874141)

摘要/Abstract

摘要：

多肽作为神经递质、激素、受体等参与和调节许多重要的生命过程。随着对多肽结构与功能认识的深入，化学合成多肽在生物医药、生命分析等领域显示了重要价值。人心房钠尿肽（ANP）是一种由28个氨基酸组成的多肽类激素，其含量和代谢异常与心血管疾病、癌症等密切相关。针对ANP重要的生理功能，该文开展了ANP化学合成及其过程的监测与优化，设计了集固相合成线性直链多肽和液相氧化成环为一体的策略。基于高效液相色谱的分离性能和多级质谱的结构鉴定能力，建立了超高效液相色谱-串联质谱（UHPLC-MS/MS）分析新方法，对固相合成产物进行了分离分析，并对液相氧化方法进行了筛选和优化，最终分离纯化得到了目标多肽，为人心房钠尿肽的高效化学合成及氧化成环反应提供借鉴。

关键词: 超高效液相色谱-质谱联用, 多肽固相合成, 人心房钠尿肽, 氧化反应

Abstract:

Peptides participate in and regulate many important life processes as neurotransmitters, hormones and receptors. With detailed understanding of the structure and function of peptides, chemically synthesized peptides have shown great value in the fields of biomedicine and life analysis. Human atrial natriuretic peptide (ANP) is a polypeptide hormone consisting of 28 amino acids. The abnormalities in the content and metabolism of ANP are closely related to the diseases like cardiovascular diseases and cancer. In view of the important physiological functions of ANP, herein we monitored and optimized the chemical synthesis of ANP chemical synthesis. The strategy involved integrating solid phase synthesis of linear peptide chain and liquid phase oxidation. Based on the excellent separation performance of HPLC and the excellent qualitative ability of mass spectrometry (MS), a new method based on ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was established to monitor the synthesis reaction and to separate and purify the obtained products. This study provides a reference for the efficient chemical synthesis and oxidative ring formation of human atrial natriuretic peptide.

Key words: ultra high performance liquid chromatography-mass spectrometry (UHPLC-MS), solid phase peptide synthesis (SPSS), human atrial natriuretic peptide (ANP), oxidation

徐坤, 金钰龙, 黄嫣嫣, 赵睿. 超高效液相色谱-质谱联用法监测人心房钠尿肽的化学合成[J]. 色谱, 2020, 38(3): 324-331.

XU Kun, JIN Yulong, HUANG Yanyan, ZHAO Rui. Monitoring chemical synthesis of human atrial natriuretic peptide by ultra high performance liquid chromatography-mass spectrometry analysis[J]. Chinese Journal of Chromatography, 2020, 38(3): 324-331.

图/表 7

图1 ANP合成流程图

Fig. 1 Scheme of human atrial natriuretic peptide (ANP) synthesis AA: amino acid; tBU: tert-butyl; Pbf: 2, 2, 4, 6, 7-pentamethyldihydrobenzofuran-5-sulphonyl; Trt: trityl.

图2 (a) ANP直链粗品和(b)纯化后ANP直链的RP-HPLC图

Fig. 2 RP-HPLC chromatograms of (a) crude linear ANP and (b) purified linear ANP

图3 (a) 副产物27肽和(b)ANP直链多肽的MALDI-FT-ICR质谱图

Fig. 3 MALDI-FT-ICR (ion cyclotron resonance) mass spectra of (a) by-product and (b) linear ANP

图4 ANP成环反应(碘氧化)的RP-HPLC图

Fig. 4 Chromatogram of ANP cyclization by I2 oxidation Peak Nos.: 1. by-product 1; 2. target ANP; 3. by-product 2.

图5 ANP成环反应(DMSO氧化)的HPLC色谱图和ESI-IT质谱图

Fig. 5 HPLC chromatograms and ESI-IT mass spectra of ANP cyclization by DMSO oxidation a. Chromatograms of reaction mixture at 0, 3, 6, and 8 h; b. ESI-IT (ion trap) mass spectra of linear ANP before oxidation; c. ESI-IT mass spectra of ANP after oxidation.

图6 (a) 纯化前ANP粗品和(b)纯化后ANP的RP-HPLC图

Fig. 6 RP-HPLC chromatograms of (a) the crude ANP and (b) the purified ANP

图7 ANP的(a) ESI-IT质谱图和(b和c)ESI-IT MS/MS碎片离子图

Fig. 7 (a) ESI-IT mass spectrum and (b and c) ESI-IT-MS/MS spectra of purified ANP

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超高效液相色谱-质谱联用法监测人心房钠尿肽的化学合成

Monitoring chemical synthesis of human atrial natriuretic peptide by ultra high performance liquid chromatography-mass spectrometry analysis

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