Study of a novel agent for TCA precipitated proteins washing - comprehensive insights into the role of ethanol/HCl on molten globule state by multi-spectroscopic analyses.

Sample preparation for mass spectrometry-based proteomics is a key step for ensuring reliable data. 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 difficult to 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. 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 a biofilm, a soil or a mouse liver was demonstrated (data available via ProteomeXchange with identifier PXD008110). Being versatile and simple, it could be of great interest to include an ethanol/HCl wash-step to produce high-quality protein extracts. SIGNIFICANCE: In mass spectrometry-based proteomics workflows, proteins precipitation and/or washing usually involves the use of acetone. In fact, this solvent is effective for removing both biological interferences (e.g. lipids) and chemicals employed in protein extraction/purification protocols (e.g. TCA, SDS). However, the use of acetone can lead to significant protein losses. Moreover, when proteins are precipitated with TCA, the acetone-treated precipitate remains hard to disperse, leading to poor resolubilization of proteins in aqueous buffers. Here, we investigated the use of ethanol/HCl for washing TCA-precipitated proteins, with the aim to produce high-quality protein extracts which can be directly analyzed by LC-MS. An opening study on standard solutions showed that ethanol/HCl led to reduced losses of proteins compared to usual solvents (i.e. acetone and ethanol). This reagent also enabled a better solubilization of proteins in aqueous buffer that is necessary for their direct trypsin digestion and LC-HRMS analysis. A mechanistic study, performed through several spectroscopic analyses (LC-HRMS, Raman, spectrofluorometry), showed that treatment with ethanol/HCl induced conformational changes of TCA-precipitated proteins. Finally, we compared the efficiency of ethanol/HCl to published protocols for the washing of protein extracts from three different complex samples (i.e. soil, biofilm, and mouse liver). Our results demonstrated that ethanol/HCl is a valuable alternative to previous protein washing methods and, therefore could become a useful tool in mass spectrometry-based proteomics workflows for various applications (e.g. clinical research, chemical biology, environmental metaproteomics…).