Chronopotentiometric sensing of anterior gradient 2 protein

Abstract

The study of new proteins and particularly those involved in cancer development is still a focal point of interest. We studied the anterior gradient 2 (AGR2) oncoprotein, aberrantly expressed in a number of human cancers. For the first time the electrochemical behaviour of various variants of the AGR2 was described using constant current chronopotentiometric stripping (CPS) analysis at mercury electrodes. In the first part, we show that mutation of AGR2 protein at its sole cysteine significantly changed its CPS response compared to wild type AGR2, probably due to their different adsorption and some deviations in their structures, which were obtained by analysis of hydrogen − deuterium (H/D) exchange connected with high resolution mass spectrometry (MS). In the second part we studied the influence of His-tag modification, widely used in the purification of recombinant proteins, on CPS response and H/D exchange. Addition of a His-tag, containing positively charged and electroactive residues, affected the CPS peak H of His6-tagged AGR2 compared to non-tagged AGR2 protein due to different adsorption as well as variation in the structure at the negatively charged interface. H/D exchange MS analysis confirmed differences in the structure of these two variants even in solution. The uncovering of AGR2 behaviour at charged surfaces gives us the opportunity to study its interactions with other biomedically important proteins.