Endogenous Plasma Peptide Detection and Identification in the Rat by a Combination of Fractionation Methods and Mass Spectrometry

Mass spectrometry-based analyses are essential tools in the field of biomarker research. However, detection and characterization of plasma low abundance and/or low molecular weight peptides is challenged by the presence of highly abundant proteins, salts and lipids. Numerous strategies have already been tested to reduce the complexity of plasma samples. The aim of this study was to enrich the low molecular weight fraction of rat plasma. To this end, we developed and compared simple protocols based on membrane filtration, solid phase extraction, and a combination of both. As assessed by UV absorbance, an albumin depletion >99% was obtained. The multistep fractionation strategy (including reverse phase HPLC) allowed detection, in a reproducible manner (CV < 30%–35%), of more than 450 peaks below 3000 Da by MALDI-TOF/MS. A MALDI-TOF/MS-determined LOD as low as 1 fmol/μL was obtained, thus allowing nanoLC-Chip/MS/MS identification of spiked peptides representing ~10−6% of total proteins, by weight. Signal peptide recovery ranged between 5%–100% according to the spiked peptide considered. Tens of peptide sequence tags from endogenous plasma peptides were also obtained and high confidence identifications of low abundance fibrinopeptide A and B are reported here to show the efficiency of the protocol. It is concluded that the fractionation protocol presented would be of particular interest for future differential (high throughput) analyses of the plasma low molecular weight fraction

Homodimerization of the Death-Associated Protein Kinase Catalytic Domain: Development of a New Small Molecule Fluorescent Reporter

Agence Nationale de Recherche (ANR); Centre National de la Recherche Scientifique (CNRS); University of StrasbourgBackground: Death-Associated Protein Kinase (DAPK) is a member of the Ca(2+)/calmodulin regulated serine/threonine protein kinases. Its biological function has been associated with induced cell death, and in vivo use of selective small molecule inhibitors of DAPK catalytic activity has demonstrated that it is a potential therapeutic target for treatment of brain injuries and neurodegenerative diseases. Methodology/Principal Findings: In the in vitro study presented here, we describe the homodimerization of DAPK catalytic domain and the crucial role played by its basic loop structure that is part of the molecular fingerprint of death protein kinases. Nanoelectrospray ionization mass spectrometry of DAPK catalytic domain and a basic loop mutant DAPK protein performed under a variety of conditions was used to detect the monomer-dimer interchange. A chemical biological approach was used to find a fluorescent probe that allowed us to follow the oligomerization state of the protein in solution. Conclusions/Significance: The use of this combined biophysical and chemical biology approach facilitated the elucidation of a monomer-dimer equilibrium in which the basic loop plays a key role, as well as an apparent allosteric conformational change reported by the fluorescent probe that is independent of the basic loop structure

Prise en charge de l'hypothyroïdie et iatrogènie médicamenteuse

PARIS-BIUP (751062107) / SudocSudocFranceF

Biochar modifies soil physical properties mostly through changes in soil structure rather than through its internal porosity

Besides its carbon sequestration potential, biochar application generally improves soil physical properties, but the magnitude of its impact and the underlying mechanisms remain debated and depend on soil type, biochar application rate, and age. The objective was therefore to determine the effect of biochar application rate and age on physical properties of agricultural soils in a temperate climate. On a silt loam and a sandy loam soils, we compared the physical properties of fresh biochar (1% and 2% w/w) or century-old biochar (0.5%–1% w/w; 19th-century kiln sites)-enriched soil samples with biochar-free soil samples. Biochar pore network characteristics were determined using helium pycnometry, mercury intrusion porosimetry, scanning electron microscopy observation, and electron dispersive X-ray spectrometry, whereas location of biochar particles within soil structure was analyzed using optical microscopy observations. Fresh biochar application decreased bulk density by 16.8% and increased saturated water content by 16.0% and macroporosity by 78.8%. These effects were attributed to soil structure improvement rather than to biochar porosity. Soil type and biochar application rate had a limited impact. In the long-term, biochar effects were mostly nonsignificant, which might result from its fairly low content in kiln sites and from the clogging of its internal porosity by clay particles. Biochar was thus able to improve some soil physical properties in the short-term, but these effects could no longer be detected in the very long-term. Further investigating the time rate of change in soil physical properties over several decades following biochar additions to soil would therefore seem particularly relevant

An optimized MALDI mass spectrometry method for improved detection of lysine/arginine/histidine free peptides.

International audienceTranscription factors and their regulators possess "basic amino acid free domains" which modulate transcriptional gene activation. We aimed at optimizing a MALDI mass spectrometry (MS) analytical method for the characterization of such domains after protein enzymatic digestion. A panel of recombinant transcription factors with different basic residue contents was proteolytically digested with the Asp-N endoprotease and resulting peptide mixtures were analyzed by MALDI-MS with alpha-cyano-4-hydroxy-cinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) as matrix. We found that peptides without lysine, arginine, histidine (Lys/Arg/His free peptides) were efficiently detected in the positive ion mode only when using DHB. These findings proved to be very useful for two different targeted proteomic applications. Indeed, the MALDI-MS/MS identification of the CARM1 proteolytic cleavage site, which happens in a Lys/Arg/His free domain, could only be achieved using the DHB matrix. Moreover, in routine proteomic analyses, the detection efficiency of Lys/Arg/His free C-terminal peptides of two-dimensional gel separated proteins was strongly enhanced when DHB was used instead of CHCA

New actors in regulation of HIV-1 mRNA production

International audienceRegulation of the retroviral protein production largely depends upon regulation of the alternative splicing of the viral transcript. The Tat protein is required for transcription of full-length HIV-1 RNA and therefore is essential for virus multiplication. However, as the Tat protein has apoptotic properties, virus HIV-1 limits tat mRNA production through a strong down regulation of splicing at site A3, an HIV-1 splicing site specifically dedicated to tat mRNA production. Splicing at this site is modulated by a complex array of silencer and enhancer elements contained in a long stem-loop structure SLS3, which is located downstream from site A3. Earlier studies have shown that the main inhibitory element ESS2 is a strong hnRNP A1 entry site, allowing further hnRNP A1 binding on the SLS3 region and leading to limitation of the U2AF splicing factor association. The SR proteins SC35 and SRp40 binding sites overlap ESS2, and their association strongly activates splicing at site A3. We found that two other SR proteins, ASF/SF2 and 9G8 also activate site A3 utilization. However, our experimental study revealed different pattern of interaction of these proteins with SLS3, as compared to SC35 and SRp40. They have short juxtaposed binding sites that overlap a C-to-U mutation position 5396 which was shown to induce a strong decrease of tat1 mRNA production in cellulo. The analysis by mass spectrometry of the proteins bound on SLS3 with a C or a U residue at position 5396 revealed the presence of the inhibitory splicing protein DAZAP1. Its binding is strongly reinforced by the C to U substitution. Based on our present data, the activation properties of ASF/SF2 and 9G8 proteins on site A3 are based on their capability to block DAZAP1 binding to SLS3. Taken together, the data reveal that numerous nuclear factors of the infected cells are involved in the complex regulation of HIV-1 RNA splicing

New actors in regulation of HIV-1 mRNA production

International audienceRegulation of the retroviral protein production largely depends upon regulation of the alternative splicing of the viral transcript. The Tat protein is required for transcription of full-length HIV-1 RNA and therefore is essential for virus multiplication. However, as the Tat protein has apoptotic properties, virus HIV-1 limits tat mRNA production through a strong down regulation of splicing at site A3, an HIV-1 splicing site specifically dedicated to tat mRNA production. Splicing at this site is modulated by a complex array of silencer and enhancer elements contained in a long stem-loop structure SLS3, which is located downstream from site A3. Earlier studies have shown that the main inhibitory element ESS2 is a strong hnRNP A1 entry site, allowing further hnRNP A1 binding on the SLS3 region and leading to limitation of the U2AF splicing factor association. The SR proteins SC35 and SRp40 binding sites overlap ESS2, and their association strongly activates splicing at site A3. We found that two other SR proteins, ASF/SF2 and 9G8 also activate site A3 utilization. However, our experimental study revealed different pattern of interaction of these proteins with SLS3, as compared to SC35 and SRp40. They have short juxtaposed binding sites that overlap a C-to-U mutation position 5396 which was shown to induce a strong decrease of tat1 mRNA production in cellulo. The analysis by mass spectrometry of the proteins bound on SLS3 with a C or a U residue at position 5396 revealed the presence of the inhibitory splicing protein DAZAP1. Its binding is strongly reinforced by the C to U substitution. Based on our present data, the activation properties of ASF/SF2 and 9G8 proteins on site A3 are based on their capability to block DAZAP1 binding to SLS3. Taken together, the data reveal that numerous nuclear factors of the infected cells are involved in the complex regulation of HIV-1 RNA splicing

Analysis of recombinant phosphoprotein complexes with complementary mass spectrometry approaches

The baculovirus expression vector system is recognized as a powerful and versatile tool for producing large quantities of recombinant proteins that cannot be obtained in Escherichia coli. Here we report (i) the purification of the recombinant cyclin-dependent kinase (CDK)-activating kinase (CAK) complex, which includes CDK7, cyclin H, and MAT1 proteins, and (ii) the functional characterization of CAK together with a detailed analysis and mapping of the phosphorylation states and sites using mass spectrometry (MS). In vitro kinase assay showed that recombinant CAK is able to phosphorylate the cyclin-dependent kinase CDK2 implicated in cell cycle progression and the carboxy-terminal domain (CTD) of the eukaryotic RNA polymerase II. An original combination of MS techniques was used for the determination of the phosphorylation sites of each constitutive subunit at both protein and peptide levels. Liquid chromatography (LC)-MS analysis of intact proteins demonstrated that none of the CAK subunits was fully modified and that the phosphorylation pattern of recombinant CAK is extremely heterogeneous. Finally, matrix-assisted laser desorption/ionization (MALDI)-MS and nanoLC-tandem mass spectrometry (MS/MS) techniques were used for the analysis of the major phosphorylation sites of each subunit, showing that all correspond to Ser/Thr phosphorylation sites. Phosphorylations occurred on Ser164 and Thr170 residues of CDK7, Thr315 residue of cyclin H, and Ser279 residue of MAT1