TCA precipitation and ethanol/HCl single-step purification evaluation: One-dimensional gel electrophoresis, bradford assays, spectrofluorometry and Raman spectroscopy data on HSA, Rnase, lysozyme - Mascots and Skyline data

The data presented here are related to the research paper entitled “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” (Eddhif et al., submitted for publication) [1]. The suitability of ethanol/HCl for the washing of TCA-precipitated proteins was first investigated on standard solution of HSA, cellulase, ribonuclease and lysozyme. Recoveries were assessed by one-dimensional gel electrophoresis, Bradford assays and UPLC-HRMS. The mechanistic that triggers protein conformational changes at each purification stage was then investigated by Raman spectroscopy and spectrofluorometry. Finally, the efficiency of the method was evaluated on three different complex samples (mouse liver, river biofilm, loamy soil surface). Proteins profiling was assessed by gel electrophoresis and by UPLC-HRMS.

Finally, the efficiency of the method was evaluated on three different complex samples (mouse liver, river biofilm, loamy soil surface). Proteins profiling was assessed by gel electrophoresis and by UPLC Proteins extraction was performed on 500 mg of soil, 10 mg of biofilm and 15 mg of mouse liver as starting material according to protocols of Chourey et al. [2], Huang et al. [3] and Song et al. [4] respectively. Proteins were precipitated with 25% (w/v) trichloroacetic acid (TCA). The washing of protein pellet was performed with three different agents (acetone, ethanol, or ethanol/HCl). The mixture was vortexed and kept at −20°C for 1 h, centrifuged at 16,600 g for 15 min at 4°C. The resulting pellets were dried in a SpeedVac concentrator, solubilized in a 50 mM of ammonium bicarbonate buffer containing 10 mM of Tris. Proteins were subjected to trypsin digestion for 24 h at 37°C. Digestion was stopped with formic acid before gel, bradford and mass analysis.

Poitiers, France
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Value of the data
Data show a comprehensive evaluation of protein conformational changes throughout TCA precipitation and one single step purification with various solvents.
Data highlight the efficiency of ethanol/HCl purification for TCA-precipitated proteins.
Ethanol/HCl represents a quick and inexpensive purification agent for proteomics studies. Presence and variability of proteins are potential values to determine which purification method must be used for proteomics investigation.

Data
TCA precipitation is one of the most common and robust technique required for protein analyses [5][6][7]. However it leads to molten globule states which hamper the solubilization of proteins in aqueous buffers for mass spectrometry analysis.

Effects of successive ethanol/HCl washings on proteins recoveries
10 mg of biofilm samples were spiked with the standard solution of HSA, exoglucanase 1 from the mix of cellulase, lysozyme, and ribonuclease A (Rnase). Proteins final concentration was 1 µg mg −1 of matrix to enable HRMS detection of the proteins after the whole process. The mixture was vortexed and left during 24 h at room temperature to favor proteins adsorption on the matrix. After extraction following the published protocol of Huang et al. [3], protein pellets were subjected to one, two or three ethanol/HCl washing(s).
They were then dissolved in 50 mM of ammonium bicarbonate containing 10 mM of Tris (pH 8.5), diluted in a ratio of 1:3 using the same buffer and subjected to trypsin digestion. Experiments were performed in triplicate. Fig. 3 gives the mean protein recoveries following the designed approach (Ethanol/HCl) on biofilm matrix after multiple washing steps. To get insights into the role of ethanol/HCl on proteins solubility, their conformational changes were comprehensively investigated, as an extension of the results reported in Ref. [1]. These measures were performed at each purification stage with two spectroscopic techniques: spectrofluorometry and Raman.

Published approach-mouse liver Published approach-biofilm
Designed approach-mouse liver Published approach-soil Designed approach-biofilm Designed approach-soil

Raman microspectroscopy
Raman spectrum for Rnase, is presented in Fig. 7. Spectra and curve fitting of the amide I band of proteins corresponding to lysozyme and HSA are presented in Figs. 5 and 6 in Ref. [1], respectively (Figs. 8-11). The unfolding or aggregation of proteins usually involves some dynamic changes in their secondary structures. These changes are mainly monitored by the analysis of the amide I region (1600-1690 cm −1 ) which is assumed to be sensitive to α-helical secondary structures [8].

Extraction and purification of proteins from complex samples: LC-HRMS analysis
We present processed data of UPLC-HRMS analysis of proteins from different samples (mouse liver, river biofilm, soil) after TCA precipitation and solvent purification. The datasets in XML format  can be used to evaluate ethanol/HCl purification for proteins profiling. Table 1 gives the HRMS features of peptides targeted for the standard proteins after in silico tryptic digestion. Table 2 presents endogenous proteins identified in soil, biofilm and mouse liver samples after purification following either the designed approach or published protocols (Mascot identification). Table 3 presents endogenous proteins detected in the mouse liver sample and quantified through Skyline with corresponding peptides and transitions for PRM. Table 4 presents endogenous proteins detected in the biofilm sample and quantified through Skyline with corresponding peptides and transitions for PRM (Table 5).     11. Relative integrated intensities of HSA amide I contribution from peak #1 assigned to unordered structures (uo), peak#2 (ordered α helices, ho), peak#3 (unordered α helices and β sheets, hu þ sh), and peak #4 (turns, tu) as obtained after profile fitting of amide I region of the Raman spectra shown in Fig. 6 [1]. Values on top of each bar correspond to the Raman shift on which the contribution peak was centred at the end of the fitting.       Table 5 Total spectrum, peptide and protein counts after purification by our approach versus published protocols on complex matrices.

Experimental design, materials and methods
Experimental design and materials and methods have been reported previously [1].