MALDI-TOF/MS-based label-free binding assay for angiotensin II type 1 receptor: application for novel angiotensin peptides.
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| Abstract |
Angiotensin (Ang) II is critically involved in multiple cardiovascular diseases mainly through the Ang II type 1 (AT1) receptor. Thus, AT1 receptor antagonists have been widely used for the treatment of hypertension and other cardiovascular conditions. However, most binding assays for AT1 receptor are based on radioactivity or fluorescent labeling, which present disadvantages such as radioactive waste production and/or changes to the binding characteristics of the labeled receptor or ligand. This article describes the first matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MS)-based label-free binding assay for AT1 receptor that can be performed in a high-throughput format. The affinity of competitors can be directly compared to that of Ang II by quantifying Ang II dissociated from the receptor-ligand complex using 50% methanol in water. The sensitivity and selectivity of MS analysis was improved by employing a stable-isotope ((13)C, (15)N)-labeled internal standard. This assay provided binding results equivalent to those obtained using other methods and revealed a significant decrease in the binding affinity of Ang II when the N-terminus underwent oxidative modification. The current setup of the assay can be easily adapted for other Ang receptors and would facilitate drug discovery studies targeting Ang receptors.
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| Authors | Seon Hwa Lee, Takuro Masuda, Takaaki Goto, Tomoyuki Oe |
| Journal | Analytical biochemistry (Anal Biochem) Vol. 437 Issue 1 Pg. 10-6 (Jun 01 2013) ISSN: 1096-0309 [Electronic] United States |
| PMID | 23376574 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't) |
| Copyright | Copyright © 2013 Elsevier Inc. All rights reserved. |
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