Separation Methods hyphenated to Mass Spectrometry for the Characterization of the Protein Glycosylation at the Intact Level

International audienceGlycosylation is one of the most common post-translational modifications of proteins that affects their biological activity, solubility, and half-life. Therefore, its characterization is of great interest in proteomic, particularly from a diagnostic and therapeutic point of view. However, the number and type of glycosylation sites, the degree of site occupancy and the different possible structures of glycans can lead to a very large number of isoforms for a given protein, called glycoforms. The identification of these glycoforms constitutes an important analytical challenge. Indeed, to attempt to characterize all of them, it is necessary to develop efficient separation methods associated with a sensitive and informative detection mode, such as mass spectrometry (MS). Most analytical methods are based on bottom-up proteomics, which consists in the analysis of the protein at the glycopeptides level after its digestion. Even if this approach provides essential information, including the localization and composition of glycans on the protein, it is also characterized by a loss of information on macro-heterogeneity, i.e. the nature of the glycans present on a given glycoform. The analysis of glycoforms at the intact level can overcome this disadvantage. The aim of this review is to detail the state-of-the art of separation methods that can be easily hyphenated with MS for the characterization of protein glycosylation at the intact level. The different electrophoretic and chromatographic approaches are discussed in detail. The miniaturization of these separation methods is also discussed with their potential applications. While the first studies focused on 2 the development and optimization of the separation step to achieve high resolution between isoforms, the recent ones are much more application-oriented, such as clinical diagnosis, quality control, and glycoprotein monitoring in formulations or biological samples

Extraction sélective de la cocaïne d'échantillons biologiques avec un polymère à empreintes moléculaires monolithique et miniaturisé et une analyse en ligne par nano-chromatographie en phase liquide

International audienceWe report the on-line coupling of a monolithic molecularly imprinted polymer to nano-liquid chromatography for the selective analysis of cocaine and its main metabolite, benzoylecgonine, in complex biological samples. After the screening of different synthesis conditions, a monolithic molecularly imprinted polymer was in situ synthesized into a 100 µm internal diameter fused-silica capillary using cocaine as template, methacrylic acid as functional monomer, and trimethylolpropane trimethacrylate as cross-linker. Scanning electron microscopy was used to assess the homogeneous morphology of the molecularly imprinted polymer and its permeability was measured. Its selectivity was evaluated by nano-liquid chromatography-ultraviolet, leading to imprinting factors of 3.2 ± 0.5 and 2.2 ± 0.3 for cocaine and benzoylecgonine, respectively, on polymers resulting from three independent syntheses, showing the high selectivity and the repeatability of the synthesis. After optimizing the extraction protocol to promote selectivity, the monolithic molecularly imprinted polymer was successfully on-line coupled with nano-liquid chromatography-ultraviolet for the direct extraction and analysis of cocaine present in spiked human plasma and saliva samples. The repeatability of the obtained extraction recovery, between 85.4-98.7% for a plasma sample spiked at 100 ng.mL-1 , was high with relative standard deviation values lower than 5.8% for triplicate analyses on each of the three independently synthesized molecularly imprinted polymers. A linear calibration range was achieved between 100 and 2000 ng mL-1 (R 2 = 0.999). Limits of quantification of 14.5 ng mL-1 and 6.1 ng mL-1 were achieved in plasma and urine samples, respectively. The very clean-baseline of the resulting chromatogram illustrated the high selectivity brought by the monolithic molecularly imprinted polymer that allows the removal of a huge peak corresponding to the elution of interfering compounds and the easy determination of the target analyte in these complex biological samples

PPERFORMANCE EVALUATION OF TWO MIPS FOR THE SPE-LC-UV DETERMINATION OF p-[18F]MPPF IN PLASMA

FP6 STRP 516984 MI-lab-on-chi

Specificity of the metabolic signatures of fish from cyanobacteria rich lakes

International audienceThe liver metabolomes of fish from cyanobacterial-dominated ponds were investigated. Cyanobacterial metabolites were only be detected in cyanobacterial dominated ponds. The metabolomes of the 2 fish species exhibit similar correlation with cyanobacteria occurrence. Correlations between the levels of some metabolites and phycocyanin or pH were observed. a b s t r a c t With the increasing impact of the global warming, occurrences of cyanobacterial blooms in aquatic ecosystems are becoming a main worldwide ecological concern. Due to their capacity to produce potential toxic metabolites, interactions between the cyanobacteria, their cyanotoxins and the surrounding freshwater organisms have been investigated during the last past years. Non-targeted metabolomic analyses have the powerful capacity to study simultaneously a high number of metabolites and thus to investigate in depth the molecular signatures between various organisms encountering different environmental scenario, and potentially facing cyanobacterial blooms. In this way, the liver metabolomes of two fish species (Perca fluviatilis and Lepomis gibbosus) colonizing various peri-urban lakes of theÎle-de-France region displaying high biomass of cyanobacteria, or not, were investigated. The fish metabolome hydrophilic fraction was analyzed by 1 H NMR analysis coupled with Batman peak treatment for the quantification and the annotation attempt of the metabolites. The results suggest that similar metabolome profiles occur in both fish species, for individuals collected from cyanobacterial blooming lakes compared to organism from non-cyanobacterial dominant environments. Overall, such environmental metabolomic pilot study provides new research perspectives in ecology and ecotoxicology fields, and may notably provide new information concerning the cyanobacteria/fish eco-toxicological interactions

DEVELOPMENT AND VALIDATION OF A METHOD FOR THE LC DETERMINATION p-[18F]MPPF IN PLASMA USING A MISPE

Peer reviewe

Organised Genome Dynamics in the Escherichia coli Species Results in Highly Diverse Adaptive Paths

The Escherichia coli species represents one of the best-studied model organisms, but also encompasses a variety of commensal and pathogenic strains that diversify by high rates of genetic change. We uniformly (re-) annotated the genomes of 20 commensal and pathogenic E. coli strains and one strain of E. fergusonii (the closest E. coli related species), including seven that we sequenced to completion. Within the ∼18,000 families of orthologous genes, we found ∼2,000 common to all strains. Although recombination rates are much higher than mutation rates, we show, both theoretically and using phylogenetic inference, that this does not obscure the phylogenetic signal, which places the B2 phylogenetic group and one group D strain at the basal position. Based on this phylogeny, we inferred past evolutionary events of gain and loss of genes, identifying functional classes under opposite selection pressures. We found an important adaptive role for metabolism diversification within group B2 and Shigella strains, but identified few or no extraintestinal virulence-specific genes, which could render difficult the development of a vaccine against extraintestinal infections. Genome flux in E. coli is confined to a small number of conserved positions in the chromosome, which most often are not associated with integrases or tRNA genes. Core genes flanking some of these regions show higher rates of recombination, suggesting that a gene, once acquired by a strain, spreads within the species by homologous recombination at the flanking genes. Finally, the genome's long-scale structure of recombination indicates lower recombination rates, but not higher mutation rates, at the terminus of replication. The ensuing effect of background selection and biased gene conversion may thus explain why this region is A+T-rich and shows high sequence divergence but low sequence polymorphism. Overall, despite a very high gene flow, genes co-exist in an organised genome

MRSA surveillance programmes worldwide : moving towards a harmonised international approach

Multinational surveillance programmes for methicillin-resistant Staphylococcus aureus (MRSA) are dependent on national structures for data collection. This study aimed to capture the diversity of national MRSA surveillance programmes and to propose a framework for harmonisation of MRSA surveillance. The International Society of Antimicrobial Chemotherapy (ISAC) MRSA Working Group conducted a structured survey on MRSA surveillance programmes and organised a webinar to discuss the programmes’ strengths and challenges as well as guidelines for harmonisation. Completed surveys represented 24 MRSA surveillance programmes in 16 countries. Several countries reported separate epidemiological and microbiological surveillance. Informing clinicians and national policy-makers were the most common purposes of surveillance. Surveillance of bloodstream infections (BSIs) was present in all programmes. Other invasive infections were often included. Three countries reported active surveillance of MRSA carriage. Method- ology and reporting of antimicrobial susceptibility, virulence factors, molecular genotyping and epidemiological metadata varied greatly. Current MRSA surveillance programmes rely upon heterogeneous data collection systems, which hampers international epidemiological monitoring and research. To harmonise MRSA surveillance, we suggest improving the integration of microbiological and epidemiological data, implementation of central biobanks for MRSA isolate collection, and inclusion of a representative sample of skin and soft-tissue infection cases in addition to all BSI cases.peer-reviewe