Optimised methods (SDS/PAGE and LC-MS) reveal deamidation in all examined transglutaminase-mediated reactions

Transglutaminases (TGs) are a family of structurally and functionally related enzymes that catalyse calcium-dependent post-translational modifications of proteins through protein-protein crosslinking, amine incorporation, or deamidation. For many years deamidation mediated by TGs was considered to be a side reaction, but recently substrate-specific deamidations have been reported. Here we describe an optimised SDS/PAGE assay for the easy and rapid monitoring of the TG reaction with small peptides. The relative proportion of deamidation to transamidation was evaluated by densitometric analysis and confirmed by nano-liquid chromatography-nano-electrospray ionisation MS. We further investigated the effect of reaction conditions on transamidation and deamidation of TG1, TG2 and blood coagulation factor XIII A-subunit (FXIII-A) enzymes using a panel of glutamine-containing peptide substrates. The ratio of transamidation to deamidation was enhanced at high excess of the acyl-acceptor substrate and increasing pH. In addition, it was influenced by peptide substrates as well. Whereas deamidation was favoured at low cadaverine concentrations and acidic pH, no significant effect of calcium was observed on the ratio of transamidation/deamidation. Under our experimental conditions, deamidation always occurred in vitro even at high excess of the acyl-acceptor substrate, and the reaction outcome was shifted to deamidation at neutral pH. Our results provide clear evidence of the deamidation in the TG reaction, and may serve as an important approach for in vivo analysis of deamidation to better understand the role of TGs in biological events

Optimised methods (SDS/PAGE and LC-MS) reveal deamidation in all examined transglutaminase-mediated reactions

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First systematic MALDI/ESI Mass Spectrometry Comparison to Characterize Polystyrene Synthesized by Different Controlled Radical Polymerizations

International audienc

First MALDI-TOF Mass Spectrometry of Vinylidene Fluoride Telomers Endowed with Low Defect Chaining

International audienceThe radical telomerization of vinylidene fluoride (or 1,1-difluoroethylene, VDF) with iodotrifluoromethane initiated by tert-butyl peroxypivalate is presented. The VDF telomers obtained were characterized by 1H and 19F NMR spectroscopy and by matrix-assisted laser desorption−ionization time-of-flight mass spectrometry (MALDI TOF MS) that revealed low polydispersity indexes showing a pseudo-living character of this reaction regarded as an iodine transfer polymerization process. CF3 and CF2I end groups appear as suitable labels in the 19F NMR spectra to assess the number-average molar masses of these VDF telomers (n). Both these techniques showed a good agreement in terms of degree of telomerization, and the first examples of MALDI−TOF mass spectra of fluorotelomers are presented. Interestingly, all telomers obtained exhibit the structure CF3CH2CF2(VDF)nI, showing that (i) the trifluoromethyl radical preferentially attacked the methylene site of VDF with a high regioselectivity; (ii) the only transfer reaction arose from that to the CF3I and not from the monomer, the initiator, the solvent, or the polymer; and (iii) a very low defect of VDF chaining (0.73% regarded as the lowest one noted in the literature) was observed

Chemical preparation and structural characterization of a homogeneous series of chitin/chitosan oligomers

International audienceThe preparation of a homogeneous series of chitin/chitosan oligomers (chito-oligomers) with the same distribution of degrees of polymerization (DP) ranging from 2 to 12, but with various average degrees of N-acetylation (DA) from 0 to 90% is described. This DA-series was obtained according to a two-step chemical process involving (i) the production of a well-defined mixture of glucosamine (GlcN) oligomers obtained by acid hydrolysis of a fully N-deacetylated chitosan and after selective precipitations of the hydrolysis products, and (ii) the partial N-acetylation of the GlcN units of these oligomers from a hydro-alcoholic solution of acetic anhydride in a controlled manner. The characterization of this series of samples with different DAs by proton nuclear magnetic resonance (1H NMR) spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) allowed us to determine their average DA and identify the main oligomer structures constituting each mixture. Furthermore, MALDI-TOF MS was particularly helpful to study the distribution evolution of the diverse oligomers as a function of DA for the main DPs from 3 to 7. The modeling of these distributions by means of a binomial law displayed that the chemical N-acetylation of low DP GlcN oligomers, produced in a homogeneous medium, occurs randomly along the oligosaccharide chains in accordance with a statistical (Bernoullian) arrangement. In this case, the relative proportion of each chito-oligomer present in the mixture can be estimated precisely as a function of DA considering oligomers of same DP

Comparison of extracellular matrix enrichment protocols for the improved characterization of the skin matrisome by mass spectrometry

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Taking Advantage of Oxidation to Characterize Thiol-Containing Polymer Chains by MALDI-TOF Mass Spectrometry

International audienceMALDI-TOF mass spectrometry analyses revealed the oxidation of thiol-containing polymer chain-ends during sample preparation using THF as solvent. In these conditions, the extent of oxidation was hardly reproducible, and led to various types of oxidized compounds. Preparing the samples at the last minute using commercial THF stabilized with an antioxidant led to more reproducible results, with the least oxidation. However, it is demonstrated herein that thiol oxidation can be advantageously taken into profit to further ascertain the presence of the thiol at the polymer chain-end. To force thiol oxidation we used THF without any antioxidant stabilizer, thus more prone to form peroxides. Thiol-containing polymer chains can thereby be indirectly evidenced by the formation of oxidation products such as chain−chain disulfide bonds and sulfonic acid chains-ends. More importantly, in these oxidizing conditions and in the negative mode, sulfonic acid-terminated polymer chains can be more sensitively detected than thiol ones (the low pK a of sulfonic acids facilitating their anionization in MALDI source). In conclusion, performing MALDI-TOF mass spectrometry analyses in oxidizing conditions, as complement to regular analyses, was found to be very useful for the chain-end identification of different thiol-containing polymer chains

Role of AcrAB-TolC multidrug efflux pump in drug-resistance acquisition by plasmid transfer

International audienceDrug-resistance dissemination by horizontal gene transfer remains poorly understood at the cellular scale. Using live-cell microscopy, we reveal the dynamics of resistance acquisition by transfer of the Escherichia coli fertility factor–conjugation plasmid encoding the tetracycline-efflux pump TetA. The entry of the single-stranded DNA plasmid into the recipient cell is rapidly followed by complementary-strand synthesis, plasmid-gene expression, and production of TetA. In the presence of translation-inhibiting antibiotics, resistance acquisition depends on the AcrAB-TolC multidrug efflux pump, because it reduces tetracycline concentrations in the cell. Protein synthesis can thus persist and TetA expression can be initiated immediately after plasmid acquisition. AcrAB-TolC efflux activity can also preserve resistance acquisition by plasmid transfer in the presence of antibiotics with other modes of actio

Proteomic characterization of high-density lipoprotein particles from non-diabetic hemodialysis patients

Chronic kidney disease is associated with an increased cardiovascular risk, and altered biological properties of high-density lipoproteins (HDL) may play a role in these events. This study aimed to describe the HDL proteome from non-diabetic hemodialysis patients and identify potential pathways affected by the dysregulated expression of HDL proteins. HDL were sampled from nine non-diabetic hemodialysis (HD) and eight control patients. Samples were analyzed using a nano-RSLC coupled with a Q-Orbitrap. Data were processed by database searching using SequestHT against a human Swissprot database and quantified with a label-free quantification approach. Proteins that were in at least five of the eight control and six of the nine HD patients were analyzed. Analysis was based on pairwise ratios and the ANOVA hypothesis test. Among 522 potential proteins, 326 proteins were identified to be in the HDL proteome from HD and control patients, among which 10 were significantly upregulated and nine downregulated in HD patients compared to the control patients (p < 0.05). Up and downregulated proteins were involved in lipid metabolism, hemostasis, wound healing, oxidative stress, and apoptosis pathways. This difference in composition could partly explain HDL dysfunction in the chronic kidney disease (CKD) population and participate in the higher cardiovascular risk observed in this population