Measurement of the total angiotensinogen and its reduced and oxidised forms in human plasma using targeted LC-MS/MS

Dahabiyeh, Lina A.; Tooth, David; Carrell, Robin W.; Read, Randy J.; Yan, Yahui; Pipkin, Fiona Broughton; Barrett, David A.

Abstract

Angiotensinogen (AGT) is a critical protein in the renin-angiotensin-aldosterone system and may have an important role in the pathogenesis of pre-eclampsia. The disulphide linkage between cysteines 18 and 138 has a key role in the redox switch of AGT which modulates the release of angiotensin I with consequential effects on blood pressure. In this paper, we report a quantitative targeted LC-MS/MS method for the reliable measurement of the total AGT and its reduced and oxidised forms in human plasma. AGT was selectively enriched from human plasma using 2-dimensional chromatography employing Concanavalin A lectin affinity and reversed phase steps and then deglycosylated using PNGase F. A differential alkylation approach was coupled with targeted LC-MS/MS method to identify the two AGT forms in the plasma chymotryptic digest. An additional AGT proteolytic marker peptide was identified and used to measure total AGT levels. The developed MS workflow enabled the reproducible detection of total AGT and its two distinct forms in human plasma with analytical precision of ≤ 15%. The LC-MS/MS assay for total AGT in plasma showed a linear response (R2 =0.992) with a limit of quantification in the low nM range. The method gave suitable validation characteristics for biomedical application to the quantification of the oxidation level and the total level of AGT in plasma samples collected from normal and pre-eclamptic patients.

Citation

Dahabiyeh, L. A., Tooth, D., Carrell, R. W., Read, R. J., Yan, Y., Pipkin, F. B., & Barrett, D. A. (2019). Measurement of the total angiotensinogen and its reduced and oxidised forms in human plasma using targeted LC-MS/MS. Analytical and Bioanalytical Chemistry, 411(2), 427–437. https://doi.org/10.1007/s00216-018-1455-2

• Journal Article Type: Article
• Acceptance Date: Oct 25, 2018
• Online Publication Date: Nov 21, 2018
• Publication Date: 2019-01
• Deposit Date: Oct 29, 2018
• Publicly Available Date: Oct 29, 2018
• Journal: Analytical and Bioanalytical Chemistry
• Print ISSN: 1618-2642
• Electronic ISSN: 1618-2650
• Publisher: Springer Publishing Company
• Peer Reviewed: Peer Reviewed
• Volume: 411
• Issue: 2
• Pages: 427–437
• DOI: https://doi.org/10.1007/s00216-018-1455-2
• Keywords: angiotensinogen, redox switch, pre-eclampsia, LC-MS/MS, Cys18, marker peptide
• Public URL: https://nottingham-repository.worktribe.com/output/1203152
• Publisher URL: https://link.springer.com/article/10.1007%2Fs00216-018-1455-2
• Additional Information: Received: 25 July 2018; Revised: 17 October 2018; Accepted: 25 October 2018; First Online: 21 November 2018; : Human blood plasma was obtained with informed consent following approval from the School of Pharmacy Research Ethics Committee, University of Nottingham, UK.; : The authors declare that they have no conflict of interest.
• Contract Date: Oct 29, 2018