Publications by authors named "Lucrece Matheron"

16 Publications

  • Page 1 of 1

In vivo characterization of the activities of novel cyclodipeptide oxidases: new tools for increasing chemical diversity of bioproduced 2,5-diketopiperazines in Escherichia coli.

Microb Cell Fact 2020 Sep 7;19(1):178. Epub 2020 Sep 7.

Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France.

Background: Cyclodipeptide oxidases (CDOs) are enzymes involved in the biosynthesis of 2,5-diketopiperazines, a class of naturally occurring compounds with a large range of pharmaceutical activities. CDOs belong to cyclodipeptide synthase (CDPS)-dependent pathways, in which they play an early role in the chemical diversification of cyclodipeptides by introducing Cα-Cβ dehydrogenations. Although the activities of more than 100 CDPSs have been determined, the activities of only a few CDOs have been characterized. Furthermore, the assessment of the CDO activities on chemically-synthesized cyclodipeptides has shown these enzymes to be relatively promiscuous, making them interesting tools for cyclodipeptide chemical diversification. The purpose of this study is to provide the first completely microbial toolkit for the efficient bioproduction of a variety of dehydrogenated 2,5-diketopiperazines.

Results: We mined genomes for CDOs encoded in biosynthetic gene clusters of CDPS-dependent pathways and selected several for characterization. We co-expressed each with their associated CDPS in the pathway using Escherichia coli as a chassis and showed that the cyclodipeptides and the dehydrogenated derivatives were produced in the culture supernatants. We determined the biological activities of the six novel CDOs by solving the chemical structures of the biologically produced dehydrogenated cyclodipeptides. Then, we assessed the six novel CDOs plus two previously characterized CDOs in combinatorial engineering experiments in E. coli. We co-expressed each of the eight CDOs with each of 18 CDPSs selected for the diversity of cyclodipeptides they synthesize. We detected more than 50 dehydrogenated cyclodipeptides and determined the best CDPS/CDO combinations to optimize the production of 23.

Conclusions: Our study establishes the usefulness of CDPS and CDO for the bioproduction of dehydrogenated cyclodipeptides. It constitutes the first step toward the bioproduction of more complex and diverse 2,5-diketopiperazines.
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http://dx.doi.org/10.1186/s12934-020-01432-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7487605PMC
September 2020

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

Clin Res Hepatol Gastroenterol 2020 10 18;44(5):681-691. Epub 2020 Jan 18.

Assistance Publique-Hôpitaux de Paris (AP-HP), Groupe Hospitalier Pitié-Salpêtrière (GHPS), 75013 Paris, France; Sorbonne Université, Inserm, Centre de Recherche Saint-Antoine (CRSA), 75012 Paris, France. Electronic address:

Background: 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.
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http://dx.doi.org/10.1016/j.clinre.2019.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367700PMC
October 2020

Free cholesterol transfer to high-density lipoprotein (HDL) upon triglyceride lipolysis underlies the U-shape relationship between HDL-cholesterol and cardiovascular disease.

Eur J Prev Cardiol 2020 10 15;27(15):1606-1616. Epub 2019 Dec 15.

National Institute for Health and Medical Research (INSERM) UMR_S 1166, Faculty of Medicine Pitié-Salpétrière, Paris, France.

Background: Low concentrations of high-density lipoprotein cholesterol (HDL-C) represent a well-established cardiovascular risk factor. Paradoxically, extremely high HDL-C levels are equally associated with elevated cardiovascular risk, resulting in the U-shape relationship of HDL-C with cardiovascular disease. Mechanisms underlying this association are presently unknown. We hypothesised that the capacity of high-density lipoprotein (HDL) to acquire free cholesterol upon triglyceride-rich lipoprotein (TGRL) lipolysis by lipoprotein lipase underlies the non-linear relationship between HDL-C and cardiovascular risk.

Methods: To assess our hypothesis, we developed a novel assay to evaluate the capacity of HDL to acquire free cholesterol (as fluorescent TopFluor® cholesterol) from TGRL upon in vitro lipolysis by lipoprotein lipase.

Results: When the assay was applied to several populations markedly differing in plasma HDL-C levels, transfer of free cholesterol was significantly decreased in low HDL-C patients with acute myocardial infarction (-45%) and type 2 diabetes (-25%), and in subjects with extremely high HDL-C of >2.59 mmol/L (>100 mg/dL) (-20%) versus healthy normolipidaemic controls. When these data were combined and plotted against HDL-C concentrations, an inverse U-shape relationship was observed. Consistent with these findings, animal studies revealed that the capacity of HDL to acquire cholesterol upon lipolysis was reduced in low HDL-C apolipoprotein A-I knock-out mice and was negatively correlated with aortic accumulation of [H]-cholesterol after oral gavage, attesting this functional characteristic as a negative metric of postprandial atherosclerosis.

Conclusions: Free cholesterol transfer to HDL upon TGRL lipolysis may underlie the U-shape relationship between HDL-C and cardiovascular disease, linking HDL-C to triglyceride metabolism and atherosclerosis.
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http://dx.doi.org/10.1177/2047487319894114DOI Listing
October 2020

The MPK8-TCP14 pathway promotes seed germination in Arabidopsis.

Plant J 2019 11 28;100(4):677-692. Epub 2019 Sep 28.

Sorbonne Université, CNRS UMR7622, Institut de Biologie Paris-Seine-Laboratoire de Biologie du Développement (IBPS-LBD), 75005, Paris, France.

The accurate control of dormancy release and germination is critical for successful plantlet establishment. Investigations in cereals hypothesized a crucial role for specific MAP kinase (MPK) pathways in promoting dormancy release, although the identity of the MPK involved and the downstream events remain unclear. In this work, we characterized mutants for Arabidopsis thaliana MAP kinase 8 (MPK8). Mpk8 seeds presented a deeper dormancy than wild-type (WT) at harvest that was less efficiently alleviated by after-ripening and gibberellic acid treatment. We identified Teosinte Branched1/Cycloidea/Proliferating cell factor 14 (TCP14), a transcription factor regulating germination, as a partner of MPK8. Mpk8 tcp14 double-mutant seeds presented a deeper dormancy at harvest than WT and mpk8, but similar to that of tcp14 seeds. MPK8 interacted with TCP14 in the nucleus in vivo and phosphorylated TCP14 in vitro. Furthermore, MPK8 enhanced TCP14 transcriptional activity when co-expressed in tobacco leaves. Nevertheless, the stimulation of TCP14 transcriptional activity by MPK8 could occur independently of TCP14 phosphorylation. The comparison of WT, mpk8 and tcp14 transcriptomes evidenced that whereas no effect was observed in dry seeds, mpk8 and tcp14 mutants presented dramatic transcriptomic alterations after imbibition with a sustained expression of genes related to seed maturation. Moreover, both mutants exhibited repression of genes involved in cell wall remodeling and cell cycle G1/S transition. As a whole, this study unraveled a role for MPK8 in promoting seed germination, and suggested that its interaction with TCP14 was critical for regulating key processes required for germination completion.
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http://dx.doi.org/10.1111/tpj.14461DOI Listing
November 2019

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

Anal Chem 2019 07 28;91(14):9102-9110. Epub 2019 Jun 28.

Sorbonne Université , École normale supérieure, PSL University, CNRS, Laboratoire des Biomolécules, LBM , 75005 Paris , France.

Affinity photo-cross-linking coupled to mass spectrometry, 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 Δ), 18:1 ( Δ) PG, and DLoPG (18:2 Δ), 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 Paternò-Büchi oxetane formation followed or not by fragmentation (retro-Paternò-Büchi) 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.
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http://dx.doi.org/10.1021/acs.analchem.9b01584DOI Listing
July 2019

Physiological effects caused by microcystin-producing and non-microcystin producing Microcystis aeruginosa on medaka fish: A proteomic and metabolomic study on liver.

Environ Pollut 2018 Mar 21;234:523-537. Epub 2017 Dec 21.

UMR 7245 CNRS/MNHN Molécules de communication et adaptation des microorganismes, Sorbonne Universités, Muséum National d'Histoire Naturelle, 12 rue Buffon, F-75231 Paris Cedex 05, France. Electronic address:

Cyanobacterial blooms have become a common phenomenon in eutrophic freshwater ecosystems worldwide. Microcystis is an important bloom-forming and toxin-producing genus in continental aquatic ecosystems, which poses a potential risk to Human populations as well as on aquatic organisms. Microcystis is known to produce along with various bioactive peptides, the microcystins (MCs) that have attracted more attention notably due to their high hepatotoxicity. To better understand the effects of cyanobacterial blooms on fish, medaka fish (Oryzias latipes) were sub-chronically exposed to either non-MC-producing or MC-producing living strains and, for this latter, to its subsequent MC-extract of Microcystis aeruginosa. Toxicological effects on liver have been evaluated through the combined approach of histopathology and 'omics' (i.e. proteomics and metabolomics). All treatments induce sex-dependent effects at both cellular and molecular levels. Moreover, the modalities of exposure appear to induce differential responses as the direct exposure to the cyanobacterial strains induce more acute effects than the MC-extract treatment. Our histopathological observations indicate that both non-MC-producing and MC-producing strains induce cellular impairments. Both proteomic and metabolomic analyses exhibit various biological disruptions in the liver of females and males exposed to strain and extract treatments. These results support the hypothesis that M. aeruginosa is able to produce bioactive peptides, other than MCs, which can induce toxicological effects in fish liver. Moreover, they highlight the importance of considering cyanobacterial cells as a whole to assess the realistic environmental risk of cyanobacteria on fish.
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http://dx.doi.org/10.1016/j.envpol.2017.11.011DOI Listing
March 2018

Deep conservation of bivalve nacre proteins highlighted by shell matrix proteomics of the Unionoida Elliptio complanata and Villosa lienosa.

J R Soc Interface 2017 01;14(126)

UMR 6282 CNRS/uB Biogéosciences, Université de Bourgogne Franche-Comté (UB-FC), Dijon, France.

The formation of the molluscan shell nacre is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell-forming tissue, the mantle. This so-called 'calcifying matrix' is a complex mixture of proteins, glycoproteins and polysaccharides that is assembled and occluded within the mineral phase during the calcification process. Better molecular-level characterization of the substances that regulate nacre formation is still required. Notable advances in expressed tag sequencing of freshwater mussels, such as Elliptio complanata and Villosa lienosa, provide a pre-requisite to further characterize bivalve nacre proteins by a proteomic approach. In this study, we have identified a total of 48 different proteins from the insoluble matrices of the nacre, 31 of which are common to both E. complanata and V. lienosa A few of these proteins, such as PIF, MSI60, CA, shematrin-like, Kunitz-like, LamG, chitin-binding-containing proteins, together with A-, D-, G-, M- and Q-rich proteins, appear to be analogues, if not true homologues, of proteins previously described from the pearl oyster or the edible mussel nacre matrices, thus forming a remarkable list of deeply conserved nacre proteins. This work constitutes a comprehensive nacre proteomic study of non-pteriomorphid bivalves that has enabled us to describe the molecular basis of a deeply conserved biomineralization toolkit among nacreous shell-bearing bivalves, with regard to proteins associated with other shell microstructures, with those of other mollusc classes (gastropods, cephalopods) and, finally, with other lophotrochozoans (brachiopods).
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http://dx.doi.org/10.1098/rsif.2016.0846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5310735PMC
January 2017

An integrated omic analysis of hepatic alteration in medaka fish chronically exposed to cyanotoxins with possible mechanisms of reproductive toxicity.

Environ Pollut 2016 Dec 27;219:119-131. Epub 2016 Oct 27.

UMR 7245 MNHN/CNRS Molécules de Communication et Adaptation des Micro-organismes, Sorbonne Universités, Muséum National d'Histoire Naturelle, CP 39, 12 Rue Buffon, 75005 Paris, France. Electronic address:

Cyanobacterial blooms threaten human health as well as the population of other living organisms in the aquatic environment, particularly due to the production of natural toxic components, the cyanotoxin. So far, the most studied cyanotoxins are microcystins (MCs). In this study, the hepatic alterations at histological, proteome and transcriptome levels were evaluated in female and male medaka fish chronically exposed to 1 and 5 μg L microcystin-LR (MC-LR) and to the extract of MC-producing Microcystis aeruginosa PCC 7820 (5 μg L of equivalent MC-LR) by balneation for 28 days, aiming at enhancing our understanding of the potential reproductive toxicity of cyanotoxins in aquatic vertebrate models. Indeed, both MC and Microcystis extract adversely affect reproductive parameters including fecundity and egg hatchability. The liver of toxin treated female fish present glycogen storage loss and cellular damages. The quantitative proteomics analysis revealed that the quantities of 225 hepatic proteins are dysregulated. In particular, a notable decrease in protein quantities of vitellogenin and choriogenin was observed, which could explain the decrease in reproductive output. Liver transcriptome analysis through Illumina RNA-seq reveals that over 100-400 genes are differentially expressed under 5 μg L MC-LR and Microcystis extract treatments, respectively. Ingenuity pathway analysis of the omic data attests that various metabolic pathways, such as energy production, protein biosynthesis and lipid metabolism, are disturbed by both MC-LR and the Microcystis extract, which could provoke the observed reproductive impairment. The transcriptomics analysis also constitutes the first report of the impairment of circadian rhythm-related gene induced by MCs. This study contributes to a better understanding of the potential consequences of chronic exposure of fish to environmental concentrations of cyanotoxins, suggesting that Microcystis extract could impact a wider range of biological pathways, compared with pure MC-LR, and even 1 μg L MC-LR potentially induces a health risk for aquatic organisms.
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http://dx.doi.org/10.1016/j.envpol.2016.10.029DOI Listing
December 2016

Deep sexual dimorphism in adult medaka fish liver highlighted by multi-omic approach.

Sci Rep 2016 08 26;6:32459. Epub 2016 Aug 26.

UMR 7245 MNHN/CNRS Molécules de Communication et Adaptation des Micro-organismes, Sorbonne Universités, Muséum National d'Histoire Naturelle, Paris, France.

Sexual dimorphism describes the features that discriminate between the two sexes at various biological levels. Especially, during the reproductive phase, the liver is one of the most sexually dimorphic organs, because of different metabolic demands between the two sexes. The liver is a key organ that plays fundamental roles in various physiological processes, including digestion, energetic metabolism, xenobiotic detoxification, biosynthesis of serum proteins, and also in endocrine or immune response. The sex-dimorphism of the liver is particularly obvious in oviparous animals, as the female liver is the main organ for the synthesis of oocyte constituents. In this work, we are interested in identifying molecular sexual dimorphism in the liver of adult medaka fish and their sex-variation in response to hepatotoxic exposures. By developing an integrative approach combining histology and different high-throughput omic investigations (metabolomics, proteomics and transcriptomics), we were able to globally depict the strong sexual dimorphism that concerns various cellular and molecular processes of hepatocytes comprising protein synthesis, amino acid, lipid and polysaccharide metabolism, along with steroidogenesis and detoxification. The results of this work imply noticeable repercussions on the biology of oviparous organisms environmentally exposed to chemical or toxin issues.
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http://dx.doi.org/10.1038/srep32459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5000296PMC
August 2016

Characterization of biases in phosphopeptide enrichment by Ti(4+)-immobilized metal affinity chromatography and TiO2 using a massive synthetic library and human cell digests.

Anal Chem 2014 Aug 4;86(16):8312-20. Epub 2014 Aug 4.

Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences and The Netherlands Proteomics Centre, Utrecht University , Padualaan 8, 3584 CH, Utrecht, The Netherlands.

Outcomes of comparative evaluations of enrichment methods for phosphopeptides depend highly on the experimental protocols used, the operator, the source of the affinity matrix, and the samples analyzed. Here, we attempt such a comparative study exploring a very large synthetic library containing thousands of serine, threonine, and tyrosine phosphorylated peptides, being present in roughly equal abundance, along with their nonphosphorylated counterparts, and use an optimized protocol for enrichment by TiO2 and Ti(4+)-immobilized metal affinity chromatography (IMAC) by a single operator. Surprisingly, our data reveal that there are minimal differences between enrichment of phosphopeptides by TiO2 and Ti(4+)-IMAC when considering biochemical and biophysical parameters such as peptide length, sequence surrounding the site, hydrophobicity, and nature of the amino acid phosphorylated. Similar results were obtained when evaluating a tryptic digest of a cellular lysate, representing a more natural source of phosphopeptides. All the data presented are available via ProteomeXchange with the identifier PXD000759.
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http://dx.doi.org/10.1021/ac501803zDOI Listing
August 2014

HGF signaling regulates Claudin-3 dynamics through its C-terminal tyrosine residues.

Tissue Barriers 2013 Oct 9;1(4):e27425. Epub 2014 Jan 9.

Hubrecht Institute for Developmental Biology and Stem Cell Research and University Medical Centre Utrecht; Utrecht, The Netherlands.

The hormone HGF regulates morphogenesis and regeneration of multiple organs and increased HGF signaling is strongly associated with metastatic cancer. At the cellular level, one of the distinct effects of HGF is the de-stabilization of cell-cell junctions. Several molecular mechanisms have been shown to be involved that mostly culminate at the E-cadherin adhesion complex. One of the key determinants in HGF-driven morphological changes is the actomyosin cytoskeleton whose organization and physical parameters changes upon stimulation. Here we have investigated how HGF affects the different actomyosin-associated cell-cell junction complexes, Nectin Junctions, Adherens Junctions and Tight Junctions in MDCK cells. We find that components of all complexes stay present at cell-cell contacts until their physical dissociation. We find that at cell-cell junctions, the mobility of Claudin-3, but not that of other cell-cell adhesion receptors, is affected by HGF. This depends on tyrosine residues that likely affect PDZ-domain interactions at the C-terminal tail of Claudin-3, although their phosphorylation is not directly regulated by HGF. Thus we uncovered Claudins as novel targets of HGF signaling at cell-cell junctions.
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http://dx.doi.org/10.4161/tisb.27425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3894234PMC
October 2013

A large synthetic peptide and phosphopeptide reference library for mass spectrometry-based proteomics.

Nat Biotechnol 2013 Jun 19;31(6):557-64. Epub 2013 May 19.

Technische Universität München, Freising, Germany.

We present a peptide library and data resource of >100,000 synthetic, unmodified peptides and their phosphorylated counterparts with known sequences and phosphorylation sites. Analysis of the library by mass spectrometry yielded a data set that we used to evaluate the merits of different search engines (Mascot and Andromeda) and fragmentation methods (beam-type collision-induced dissociation (HCD) and electron transfer dissociation (ETD)) for peptide identification. We also compared the sensitivities and accuracies of phosphorylation-site localization tools (Mascot Delta Score, PTM score and phosphoRS), and we characterized the chromatographic behavior of peptides in the library. We found that HCD identified more peptides and phosphopeptides than did ETD, that phosphopeptides generally eluted later from reversed-phase columns and were easier to identify than unmodified peptides and that current computational tools for proteomics can still be substantially improved. These peptides and spectra will facilitate the development, evaluation and improvement of experimental and computational proteomic strategies, such as separation techniques and the prediction of retention times and fragmentation patterns.
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http://dx.doi.org/10.1038/nbt.2585DOI Listing
June 2013

Direct translocation of cell-penetrating peptides in liposomes: a combined mass spectrometry quantification and fluorescence detection study.

Anal Biochem 2013 Jul 20;438(1):1-10. Epub 2013 Mar 20.

UMR 7203 CNRS, ENS, Laboratoire des Biomolécules, UPMC-Université Paris 06, 75005 Paris, France.

Cell-penetrating peptides (CPPs) can cross cell membranes in a receptor-independent manner. Two main routes for their cellular uptake have been proposed: endocytosis and direct translocation through the cell membrane. The ability of a peptide to enter cells through direct translocation can be assessed by evaluating the amount of peptide crossing the membrane of liposomes. Most methods reported so far rely on the use of fluorescent probes, which, when attached to a CPP, often alter its physical/chemical properties. Herein, a matrix-assisted laser desorption/ionization time-of-flight MS-based method is described to quantify the amount of CPP taken up into lipid vesicles and to distinguish it from the amount that is bound or inserted in the membrane. For comparison, visualization of the uptake of the same, but fluorophore-labeled, peptides into giant vesicles and cells by fluorescence microscopy is also reported. We show that membrane charge density is an important factor for direct translocation. We also show that fluorophore-labeled peptides have a different translocation behavior and that they are more toxic to cells. Alternative methods to fluorescence, such as the one reported herein, should be favored when investigating the uptake mechanism of CPPs, as fluorescent dyes can alter short peptides' physical/chemical properties and their internalization capacities.
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http://dx.doi.org/10.1016/j.ab.2013.03.009DOI Listing
July 2013

Improving the selectivity of the phosphoric acid β-elimination on a biotinylated phosphopeptide.

J Am Soc Mass Spectrom 2012 Nov 7;23(11):1981-90. Epub 2012 Sep 7.

Laboratoire des Biomolécules, Université P et M Curie, UMR 7203 UPMC-CNRS-ENS, Paris, France.

This study aims at improving the MALDI-TOF detection of a phosphorylated peptide containing a cysteine residue by β-elimination of H(3)PO(4) hardly enriched by classical methods. The experimental conditions were optimized on this phosphopeptide (biot-pAdd) and its nonphosphorylated counterpart (biot-Add). The major side-reactions were H(2)S elimination on the cysteine residues and H(2)O elimination on the non phosphorylated serine residue of biot-Add. The former dilutes the MALDI-TOF signal for the desired species. The latter gives a product similar to what is obtained by H(3)PO(4) elimination and should prompt to caution when working with a mixture between phosphorylated and non phosphorylated peptides. Modifications on the solvent, the reaction temperature and time, the nature, and concentration of the base were made. Major improvement of the selectivity of the reaction was observed in 30 % ACN, at room temperature for 4 h. However, these optimizations are specific to these sequences and should be performed anew for different peptides. The selectivity of the reaction towards H(3)PO(4) elimination is improved, but the persistence of side-reactions renders a previous sample fractionation necessary. In these optimized conditions, the ionization enhancement is 3-fold and the detection limits for biot-pAdd are similar to biot-Add (100 fmol).
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http://dx.doi.org/10.1007/s13361-012-0467-yDOI Listing
November 2012

Sequence-dependent enrichment of a model phosphopeptide: a combined MALDI-TOF and NMR study.

Anal Chem 2011 Apr 23;83(8):3003-10. Epub 2011 Mar 23.

Université P. et M. Curie Paris 6, UMR 7203 CNRS-UPMC-ENS, 4, Place Jussieu, 75005 Paris, Ecole Normale Supérieure, Département de Chimie, 24, rue Lhomond, 75005 Paris, France.

The goal of this study was to detect and quantify by MALDI-TOF MS the phosphorylation of a peptide containing the recognition motif of the Protein Kinase C (PKC). Such model peptide can be used as a phosphorylation probe to follow intracellular kinase/phosphatase activities. This study allowed us to establish relationships between sequence specificities and affinity for TiO(2) or IMAC media. The peptide has the sequence biotin-GGGGCFRTPSFLKK-NH(2) in which the serine residue can be phosphorylated. Enrichment of the corresponding phosphopeptide, by the dedicated IMAC and TiO(2) affinity chromatography methods, proved inefficient. By combining MALDI-TOF and NMR data, we first showed that the lack of affinity of the phosphopeptide for TiO(2) was partly related to the basic property of its peptide sequence. Furthermore, the peptide shows local structuration around the P(9)- S(10) segment, with formation of a salt bridge between the guanidinium group of the R(7) side chain and the phosphate moiety. In conjunction with an inadequate position of the {biotin-G(4)} N-terminal tag, this local structure could shield the phosphate group, preventing interaction with TiO(2). To improve TiO(2) affinity, the peptide sequence was modified accordingly. The new sequences retained the biological properties while their enrichment by IMAC or TiO(2) became possible.
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http://dx.doi.org/10.1021/ac103134eDOI Listing
April 2011

MALDI TOF-TOF characterization of a light stabilizer polymer contaminant from polypropylene or polyethylene plastic test tubes.

J Mass Spectrom 2010 Jan;45(1):43-50

UPMC, 7-9 quai Saint Bernard, bâtiment A, Plateforme de Protéomique et spectrométrie de masse, IFR83, 75005 Paris, France.

Disposable plasticware such as plastic test tubes are routinely used in all proteomics laboratories. Additives in polymers are used to protect them against oxygen or ultraviolet (UV) light degradation. Hindered amine light stabilizers (HALSs) are of utmost importance in modern polyolefin (polypropylene, polyethylene) stabilization. In this article, we demonstrate that the manufacturing polymeric agent: poly-(N-beta-hydroxyethyl-2,2,6,6-tetramethyl-4-hydroxy-piperidinyl succinate), known as Tinuvin-622 or Lowilite 62, from the HALS family, leaches from laboratory polypropylene or polyethylene plastic test tubes into the standard solvents for sample preparation. The analysis of these polluted samples by matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry, in the positive mode, shows highly contaminated mass spectra, due to the high sensitivity of this technique. These contaminants have mass range and mass defect similar to those of peptides arising from the digestion of a protein in a conventional proteomics study. Therefore, they can be really harmful for proteomics studies, leading to misattributions, preventing any protein identification. In this article, an MS and MS/MS fingerprint of this pollutant is given and some pieces of advice to avoid it are proposed.
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http://dx.doi.org/10.1002/jms.1687DOI Listing
January 2010