MALDI-TOF/MS-based label-free binding assay for angiotensin II type 1 receptor: Application for novel angiotensin peptides

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 (¹³C, ¹⁵N)-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.