Ion mobility mass spectrometry enables the discrimination of positional isomers and the detection of conformers from cyclic oligosaccharides-metals supramolecular complexes

Cyclic oligosaccharides are well known to interact with various metals, able to form supramolecular complexes with distinct sizes and shapes. However, the presence of various isomers in a sample, including positional isomers and conformers, can significantly impact molecular recognition, encapsulation ability and chemical reactivity. Therefore, it is crucial to have tools for deep samples probing and correlation establishments. The emerging ion mobility mass spectrometry (IM-MS) has the advantages to be rapid and sensitive, but is still in its infancy for the investigation of supramolecular assemblies. In the herein study, it was demonstrated that IM-MS is suitable to discriminate several isomers of cyclodextrins (CD)-metals complexes, used as cyclic oligosaccharide models. In this sense, we investigated branched 6-O-α-glucosyl- or 6-O-α-maltosyl-β-cyclodextrins (G1-β-CD and G2-β-CD) and their purely cyclic isomers: CD8 (γ-CD) and CD9 (δ-CD). The corresponding collision cross section (CCS) values were deducted for the main positive singly and doubly charged species. Experimental CCS values were matched with models obtained from molecular modelling. The high mobility resolving power and resolution enabled discrimination of positional isomers, identification of various conformers and accurate relative content estimation. These results represent a milestone in the identification of carbohydrate conformers that cannot be easily reached by other approaches

Oligopeptides and copeptides of homochiral sequence, via beta-sheets, from mixtures of racemic alpha-amino acids, in a one-pot reaction in water; relevance to biochirogenesis.

International audienceAs part of our studies on the biochirogenesis of peptides of homochiral sequence during early evolution, the formation of oligopeptides composed of 14-24 residues of the same handedness in the polymerization of dl-leucine (Leu), dl-phenylalanine (Phe), and dl-valine (Val) in aqueous solutions, by activation with N, N'-carbonyldiimidazole and then initiation with a primary amine, in a one-pot reaction, was demonstrated by MALDI-TOF MS using deuterium enantio-labeled alpha-amino acids. The formation of long isotactic peptides is rationalized by the following steps occurring in tandem: (i) creation of a library of short diasteroisomeric oligopeptides containing isotactic peptides in excess in comparison to a binomial kinetics, as a result of an asymmetric induction exerted by the N-terminal residue of a given handedness; (ii) precipitation of the less soluble racemic isotactic penta- and hexapeptides in the form of beta-sheets that are delineated by homochiral rims; (iii) regio-enantiospecific chain elongation occurring heterogeneously at the beta-sheets/solution interface. Polymerization of l-Leu with l-isoleucine (Ile) or l-Phe with l- (1) N-Me-histidine yielded mixtures of copeptides containing both residues. In contrast, in the polymerization of the corresponding mixtures of l- + d-alpha-amino acids, the long oligopeptides were composed mainly from oligo- l-Leu and oligo- d-Ile in the first system and oligo- d-Phe in the second. Furthermore, in the polymerization of mixtures of hydrophobic racemic alpha-amino acids dl-Leu, dl-Val, and dl-Phe and with added racemic dl-alanine and dl-tyrosine, copeptides of homochiral sequences are most dominantly represented. Possible routes for a spontaneous "mirror-symmetry breaking" process of the racemic mixtures of homochiral peptides are presented

Protein Tyrosine Kinases and Protein Tyrosine Phosphatases Are Involved in Abscisic Acid-Dependent Processes in Arabidopsis Seeds and Suspension Cells.

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Benzophenone Photoreactivity in a Lipid Bilayer To Probe Peptide/Membrane Interactions: Simple System, Complex Information

International audienceAffinity photo-cross-linking coupled to mass spec-trometry, using benzophenone (Bzp)-functionalized peptides, was used to study the noncovalent interactions of cell-penetrating peptides and lipid membranes. Using biomimetic lipid vesicles composed of saturated and unsaturated negatively charged lipids, DMPG (14:0), DPPG (16:0), DOPG (18:1 cis Δ 9), 18:1 (trans Δ 9) PG, and DLoPG (18:2 cis Δ 9, 12), allowed observation of all the classical and less common reactivities of Bzp described in the literature by direct MS analysis: CC double bond formation on saturated fatty acids, covalent adducts formation via classical C−C bond, and Paterno-Buchi oxetane formation followed or not by fragmentation (retro-Paterno-Buchi) as well as photosensitization of unsaturated lipids leading to lipid dimers. All these reactions can occur concomitantly in a single complex biological system: a membrane-active peptide inserted within a phospholipid bilayer. We also detect oxidation species due to the presence of radical oxygen species. This work represents a noteworthy improvement for the characterization of interacting partners using Bzp photo-cross-linking, and it shows how to exploit in an original way the different reactivities of Bzp in the context of a lipid membrane. We propose an analytical workflow for the interpretation of MS spectra, giving access to information on the CPP/lipid interaction at a molecular level such as depth of insertion or membrane fluidity in the CPP vicinity. An application of this workflow illustrates the role of cholesterol in the CPP/lipids interaction

Selectivities in Adsorption and Peptidic Condensation in the (Arginine and Glutamic Acid)/Montmorillonite Clay System

The present study examines the selective adsorption and polymerization of two amino acids, glutamic acid (Glu) and arginine (Arg), on a cationic clay mineral, montmorillonite (Mt). Two experimental procedures were used: selective adsorption and wet impregnation. In the first case, an adsorption selectivity is observed based on pH-dependent speciation of the amino acids. At natural pH, arginine is positively charged and thus extensively exchanges the cations in the interlayer space of the montmorillonite whereas glutamic acid is negatively charged and adsorbed in weak amounts, probably on the clay edges. In contrast, incipient wetness impregnation forces equivalent quantities of both amino acids to be deposited. After moderate thermal activation, combined characterization techniques, especially solid-state NMR and matrix-assisted laser desorption ionization time-of-flight analysis, highlight a peptidic condensation between the amino acids and hint at a selective polymerization yielding preferably heteropeptides (e.g., cyclo­(Glu-Arg)) rather than homopeptides

Input of serum haptoglobin fucosylation profile in the diagnosis of hepatocellular carcinoma in patients with non-cirrhotic liver disease

International audienceBackground: Haptoglobin bifucosylated tetra-antennary glycan have been identified in patients with early stage hepatocellular carcinoma, but its specificity according to the presence or not of cirrhosis has never been assessed. The aims of this study were to determine if haptoglobin bifucosylated tetra-antennary glycan (1) could be a marker of HCC in patients without cirrhosis; (2) could increase the performance of standard alpha-fetoprotein (AFP) or recent blood tests for HCC detection, i.e., lectin-reactive alpha-fetoprotein (AFP-L3), des-gamma-carboxy prothrombin (DCP) and Liver-Cancer-Risk-test (LCR1-test).Methods: We retrospectively selected patients, 102 with HCC (21 without cirrhosis), matched by stages with 140 controls without HCC (81 without cirrhosis). Haptoglobin fucosylation was assessed by MALDI-TOF. LCR-glycan algorithm was constructed combining components of the LCR-1 test (haptoglobin, gammaglutamyl-transpeptidase, apolipoproteinA1, alpha-2-macroglobulin) with AFP, AFP-L3, DCP and haptoglobin bifucosylated tetra-antennary glycan.Results: In 102 patients without cirrhosis (21 HCC and 81 controls), the intention-to-diagnose analyses showed that haptoglobin bifucosylated tetra-antennary glycan alone had a sensitivity of 71% (15/21;95%CI 50-86), significantly better (P=0.02) than standard AFP (43%;9/21;95%CI 24-63), and a specificity of 96% (78/81;95% 90-99). The sensitivity of LCR-glycan, in patients without cirrhosis, was 86% (18/21; 95%CI 63-95) significantly better (P=0.001) than standard AFP (43%; 9/21; 95%CI 24-63), with an AUROC of 0.943 (95%CI 0.806-0.98) compared to 0.811 (95%CI 0.630-0.908) for AFP (P=0.06).Conclusion: Haptoglobin bifucosylated tetra-antennary glycan is associated with the presence of HCC in patients with chronic liver disease including those without cirrhosis. Its combination with existing HCC biomarkers could improve the performance of standard AFP for HCC detection

Gender-Specific Toxicological Effects of Chronic Exposure to Pure Microcystin-LR or Complex <i>Microcystis aeruginosa</i> Extracts on Adult Medaka Fish

Cyanobacterial blooms often occur in freshwater lakes and constitute a potential health risk to human populations, as well as to other organisms. However, their overall and specific implications for the health of aquatic organisms that are chronically and environmentally exposed to cyanobacteria producing hepatotoxins, such as microcystins (MCs), together with other bioactive compounds have still not been clearly established and remain difficult to assess. The medaka fish was chosen as the experimental aquatic model for studying the cellular and molecular toxicological effects on the liver after chronic exposures (28 days) to environmentally relevant concentrations of pure MC-LR, complex extracts of MC producing or nonproducing cyanobacterial biomasses, and of a <i>Microcystis aeruginosa</i> natural bloom. Our results showed a higher susceptibility of females to the different treatments compared to males at both the cellular and the molecular levels. Although hepatocyte lysis increased with MC-containing treatments, lysis always appeared more severe in the liver of females compare to males, and the glycogen cellular reserves also appeared to decrease more in the liver of females compared to those in the males. Proteomic investigations reveal divergent responses between males and females exposed to all treatments, especially for proteins involved in metabolic and homeostasis processes. Our observations also highlighted the dysregulation of proteins involved in oogenesis in female livers. These results suggest that fish populations exposed to cyanobacteria blooms may potentially face several ecotoxicological issues