Claude Geffroy-Rodier

Search for evidence of life in space: analysis of enantiomeric organic molecules by N,N-dimethylformamide dimethylacetal derivative dependant Gas Chromatography-Mass Spectrometry.

Within the context of the future space missions to Mars (MSL 2011 and Exomars 2016), which aim at searching for traces of life at the surface, the detection and quantitation of enantiomeric organic molecules is of major importance. In this work, we have developed and optimized a method to derivatize and analyze chiral organic molecules suitable for space experiments, using N,N-dimethylformamide dimethylacetal (DMF-DMA) as the derivatization agent. The temperature, duration of the derivatization reaction, and chromatographic separation parameters have been optimized to meet instrument design constraints imposed upon space experiment devices. This work demonstrates that, in addition to its intrinsic qualities, such as production of light-weight derivatives and a great resistance to drastic operating conditions, DMF-DMA facilitates simple and fast derivatization of organic compounds (three minutes at 140 degrees C in a single-step) that is suitable for an in situ analysis in space. By using DMF-DMA as the derivatization agent, we have successfully identified 19 of the 20 proteinic amino acids and been able to enantiomerically separate ten of the potential 19 (glycine being non-chiral). Additionally, we have minimized the percentage of racemized amino acid compounds produced by optimizing the conditions of the derivatization reaction itself. Quantitative linearity studies and the determination of the limit of detection show that the proposed method is also suitable for the quantitative determination of both enantiomeric forms of most of the tested amino acids, as limits of detection obtained are lower than the ppb level of organic molecules already detected in Martian meteorites.

Gas chromatography-mass spectrometry of hexafluoroacetone derivatives: First time utilization of a gaseous phase derivatizing agent for analysis of extraterrestrial amino acids

Within the perspective of the current and next space missions to Mars (MSL 2011 and Exomars 2016-2018), the detection and enantioselective separation of building blocks such as the amino acids are important subjects which are becoming fundamental for the search for traces of life on the surface and subsurface of Mars. In this work, we have developed and optimized a method adapted to space experimentation to derivatize and analyze amino acids, using hexafluoroacetone as the derivatizing agent. The temperature, duration of the derivative transfer to the analyser, and chromatographic separation parameters have been optimized to meet the instrument design constraints imposed on devices for extraterrestrial experiments. The work presented in this rationale has established that hexafluoroacetone, in addition to its intrinsic qualities, such as the production of light-weight derivatives (no racemization) and great resistance to the drastic operating conditions, has indeed facilitated simple and fast derivatization that appears to be suitable for in situ analysis in space. By using hexafluoroacetone as the derivatizing agent, we successfully identified, 21 amino acids including 12 of the 20 proteinic amino acids without stirring or extraction steps. Ten of these derivatized amino acids were enantioselectively separated. The precision and accuracy measurements for the D/L ratio showed that the proposed method was also suitable for the determination of both enantioselective forms of most of the tested amino acids. The limits of detection obtained were lower than the ppb level of organic molecules detected in Martian meteorites.

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…).

Development of liquid chromatography high resolution mass spectrometry strategies for the screening of complex organic matter: Application to astrophysical simulated materials

The present work aims at developing two LC-HRMS setups for the screening of organic matter in astrophysical samples. Their analytical development has been demonstrated on a 100-µg residue coming from the photo-thermo chemical processing of a cometary ice analog produced in laboratory. The first 1D-LC-HRMS setup combines a serially coupled columns configuration with HRMS detection. It has allowed to discriminate among different chemical families (amino acids, sugars, nucleobases and oligopeptides) in only one chromatographic run without neither a priori acid hydrolysis nor chemical derivatisation. The second setup is a dual-LC configuration which connects a series of trapping columns with analytical reverse-phase columns. By coupling on-line these two distinct LC units with a HRMS detection, high mass compounds (350 <m/z < 600) have been efficiently preconcentrated, separated and detected. Our strategies demonstrate a real interest for the analysis of astrophysical samples coming in minute quantities and allowing only few analytical runs. Besides its relevance for astrobiological studies, this work points out the suitability of these two novel LC-HRMS strategies for untargeted analysis of complex environmental samples.

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.