1D continuous gel electrophoresis composition for the separation of deamidated proteins

Abstract

Deamidation is a spontaneous modification of peptides and proteins that has potent repercussions on their activity and stability in vivo and in vitro. Being able to implement easy techniques to detect and quantify protein deamidation is a major goal in this field. Here we focus on electrophoretic methods that can be deployed to assess protein deamidation. We provide an update on the use of Taurine/Glycinate as trailing ions to assist the detection of several examples of deamidated proteins, namely the small GTPases RhoA, Rac1 and Cdc42, but also the oncogene Bcl-xL and calcium-binding Calmodulin. We also report on the use of imidazole as a counter ion to improve the focusing of deamidated bands. Finally, we provide examples of how these gels proved useful to compare on full-length proteins the effect of ions and pH on the catalytic rates of spontaneous deamidation. Taken together, the electrophoretic method introduced here proves useful to screen at once the effect of various conditions of pH, ionic strength and buffer ions on protein stability. Direct applications can be foreseen to tailor buffer formulations to control the stability of proteins drug products.

Keywords: Bcl-xL; Buffer formulation; Calmodulin; Cdc42; Denaturing electrophoresis; Protein stability; Rac1; RhoA; Separation of deamidated proteins.