Efficiency of the purification protocols on complex matrices: biofilm, mouse liver and soil

In gel-free experimental workflows, protein purification often starts with a precipitation stage using trichloroacetic acid (TCA). In presence of TCA, proteins precipitate in a stable molten globule state making the pellet difficultto solubilize in aqueous buffer for proteolytic digestion and MS analysis. In this context, the objective of this work was to study the suitability of a novel agent, ethanol/HCl, for the washing of TCA-precipitated proteins. This method optimized the recovery of proteins in aqueous buffer (50 to 96 %) while current organic solvents led to losses of material (up to 75 % of loss). Following a mechanistic study, the effect of ethanol/HCl on the conformation of TCA-precipitated proteins was investigated. It was shown that the reagent triggered the unfolding of TCA-stabilized molten globule into a reversible intermediate, characterized by a specific Raman signature, which favored protein subsequent resolubilization. Finally, the efficiency of ethanol/HCl for the washing of TCA-precipitated proteins extracted from biofilm, soil or mouse liver was demonstrated. Being versatile and simple, it could be of great interest to include an ethanol/HCl wash-step in mass spectrometry-based proteomics workflows to produce high-quality protein extracts.