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

doi:10.3724/SP.J.1123.2019.10020

Abstract

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

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.

Figures/Tables 7

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.

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

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

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

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.

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

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

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