Establishment of substance P modified affinity chromatography for specific detection and enrichment of Mas-related G protein-coupled receptor X2

Mas-related G protein-coupled receptor X2 (MrgX2) has been identified to be critical in drug-induced pseudo-allergic reactions and allergic diseases. Herein, an affinity high-performance liquid chromatog-raphy was established for the specific detection and enrichment of MrgX2. Substance P was used as an affinity ligand and immobilized on a glutaraldehyde-modified amino silica gel. The successful graft-ing of substance P was characterized by infrared spectroscopy, elemental analysis, X-ray photoelectron spectroscopy, thermogravimetric analysis, and nitrogen adsorption and desorption analyzes. The prepared materials were then used as the stationary phase to investigate the retention behavior of MrgX2 re-combinant protein on the affinity column. The results obtained with the analytical techniques show the specificity and selectivity of the MrgX2 recombinant protein on the affinity column. The repeatability and reproducibility for the analysis of MrgX2 on the NH2-Silico@GD@SP column show relative standard de-viation (RSD) values lower than the acceptance criteria of 2 and 5% of retention time, and RSD of peak areas < 7%. The RSD value of the results obtained for the control of the activity of the prepared columns respond to the acceptance criteria of 5% and proves that the NH2-Silico@GD@SP column are stable un -til 48 h. The suitability of the NH2-Silico@GD@SP column offline SEC system has been tested by using MrgX2 as positive control. The results of this experiment indicate that the offline system may be used to analyze the retention fraction. MrgX2 extracted from human mast cells LAD2 was also verified. An obvi-ous retention can be observed and the natural MrgX2 was concentrated 114.6 times compared with the original complex components by using the affinity column. These results may provide a new approach for the specific detection and enrichment of G-protein-coupled receptors. (C) 2021 Elsevier B.V. All rights reserved.

Automated summary

A novel affinity chromatography method was developed for the specific detection and enrichment of MrgX2, demonstrating the specificity and selectivity of MrgX2 recombinant protein on the affinity column. The method showed good repeatability and stability, with potential applications in the specific detection and enrichment of G-protein-coupled receptors.