Diagonal chromatographic selection of cysteinyl peptides modified with benzoquinones

The derivatization of cysteine-containing peptides with benzoquinone compounds is rapid, quantitative and specific in acidic media. The conversion of cysteines into hydrophobic benzoquinone-adducted residues in peptides is used here to alter the chromatographic properties of cysteinyl peptides during liquid chromatography separation. The benzoquinone derivatization is shown to allow the accurate selection of cysteine-containing peptides of bovine serum albumin tryptic digest by diagonal reversed-phase chromatography, which consists of one primary and a series of secondary identical liquid chromatographic separations, before and after a cysteinyl-targeted modification of the peptides by benzoquinone compounds. Figure Diagonal chromatographic selection of cysteinyl peptides modified with benzoquinone

The promise of multi-omics approaches to discover biological alterations with clinical relevance in Alzheimer's disease

Beyond the core features of Alzheimer's disease (AD) pathology, i.e. amyloid pathology, tau-related neurodegeneration and microglia response, multiple other molecular alterations and pathway dysregulations have been observed in AD. Their inter-individual variations, complex interactions and relevance for clinical manifestation and disease progression remain poorly understood, however. Heterogeneity at both pathophysiological and clinical levels complicates diagnosis, prognosis, treatment and drug design and testing. High-throughput "omics" comprise unbiased and untargeted data-driven methods which allow the exploration of a wide spectrum of disease-related changes at different endophenotype levels without focussing a priori on specific molecular pathways or molecules. Crucially, new methodological and statistical advances now allow for the integrative analysis of data resulting from multiple and different omics methods. These multi-omics approaches offer the unique advantage of providing a more comprehensive characterisation of the AD endophenotype and to capture molecular signatures and interactions spanning various biological levels. These new insights can then help decipher disease mechanisms more deeply. In this review, we describe the different multi-omics tools and approaches currently available and how they have been applied in AD research so far. We discuss how multi-omics can be used to explore molecular alterations related to core features of the AD pathologies and how they interact with comorbid pathological alterations. We further discuss whether the identified pathophysiological changes are relevant for the clinical manifestation of AD, in terms of both cognitive impairment and neuropsychiatric symptoms, and for clinical disease progression over time. Finally, we address the opportunities for multi-omics approaches to help discover novel biomarkers for diagnosis and monitoring of relevant pathophysiological processes, along with personalised intervention strategies in AD

Thiol-targeted microspray mass spectrometry of peptides and proteins through on-line EC-tagging

Modification strategies targeting specific amino acids in proteins are widespread in proteomic analysis. Cysteine residues have received deep consideration in view of their nucleophilic properties and their occurrence in the proteome. A recently developed micro-electrospray emitter for mass spectrometry was used to electrogenerate species reactive towards specific residues in biomolecules. When spraying L-cysteine in the presence of hydroquinone, the thiol cysteine moiety reacts via a 1,4-Michael addition with the benzoquinone electrochemically generated at the electrode. A series of electrogenerated selective electrophiles based on substituted benzoquinones was characterized as tags for L-cysteine. The rate constants pertaining to the addition of L-cysteine onto the benzoquinones were determined through electrochemical techniques. It was shown that the rate constants are primarily dependent on the electronic nature of the substituents. The apparent tagging extents observed for L-cysteine in microspray mass spectrometry experiments were shown to be highly dependent of the ionization efficiencies of the tag. The on-line mass spectrometric electrochemical tagging (EC-tagging) of cysteine residues was studied for peptides. The EC-tagging was tested with the different hydroquinones on an undecapeptide containing one cysteine residue. Methoxycarbonyl-1,4-hydroquinone was shown to be the most efficient probe and revealed to be suitable to count cysteine units in peptides containing up to three cysteines. The number of cysteines corresponds to the number of characteristic mass shifts observed from the unmodified peptide. The identification of bovine serum albumin and human a-lactalbumin digest samples in a peptide mapping strategy were greatly improved by the application of the EC-tagging technique as post-column treatment. Indeed, the determination of cysteine content in the tryptic peptides provides powerful supplementary information to the masses. The tagging method was applied to the determination of four proteins in a model mixture. In parallel, the microspray emitter was characterized as an electrolysis flow cell for the EC-tagging of peptides. The Levich equation was validated as a first approximation for the calculation of the convection-diffusion limiting current in the device. A finite element simulation of the multi-tagging process of peptides was developed to yield the relative distribution and concentration of tags, untagged and tagged species in the microchannel. The main chemical parameters determining the kinetics of the labelling were assessed and discussed considering the microfluidic aspects of the process. The control of the tagging extent allows the simultaneous mass spectrometric analysis of both the unmodified and of the modified peptide(s). This theoretical work has established the range of optimum conditions for the determination of the number of cysteines in peptides containing up to five cysteine groups. The mass spectrometric EC-tagging of cysteine residues in proteins was studied to probe the cysteine environment. An analytical model was developed to calculate rapidly the tagging extent before the spray event. Experiments with unmodified proteins and their chemically reduced forms have highlighted the strong effect of the cysteine site reactivity on the tagging efficiencies. This study has shown relevant parameters for such on-line electrochemical derivatization / mass spectrometric detection strategies. The chemical derivatization of cysteines by benzoquinone reagents was also investigated. These alkylating reagents revealed efficient for diagonal liquid chromatography to isolate cysteinyl peptides by the retention time shifts due to the hydrophobicity of the tags. The work has demonstrated that the inherent electrochemistry of the electrospray can be employed as post column treatment to derivatize cysteinyl biomolecules. Analytical strategies have been developed to take advantage of this electrochemically-controlled modification

Evolution du cycle hydrologique continental en France au cours des prochaines décennies

The assessment of the impact of climate change often requires to set up long chains of modeling, from the model to estimate the future concentration of greenhouse gases to the impact model. Throughout the modeling chain, sources of uncertainty accumulate making the exploitation of results for the development of adaptation strategies difficult. It is proposed here to assess impacts of climate change on the hydrological cycle over France and associated uncertainties. The contribution of each sources of uncertainty is not addressed, mainly that associated with greenhouse gases emission scenario, climate models and internal variability. In the context of impacts of climate change on the hydrological cycle over France, it is possible to ask what is the contribution of each sources of uncertainty to the total uncertainty associated with mean changes. Is it possible to reduce, and if so how, the contribution of one source or another ? We propose in this work an approach to assess the transferability in the future climate of a statistical method to downscale climate simulations. The transferability assumption is one the main sources of uncertainty in statistical downscaling method. The assessment suggested here relies on the use of regional climate models, in a perfect model framework, and shows that some predictors are useful to ensure the transferability of the downscaling method in the future climate. This framework, proposed for a statistical downscaling method, is also applicable to bias correction methods in regional climate models. Recent atmospheric reanalyses of the 20th century are downscaled with the method developed in this work, associated with observations of temperature and precipitation. The hydrological cycle over France is characterized with these reconstructions. We show that the multi-decadal variability of observed streamflows during the 20th century is generalized to the whole country and is partly due to atmospheric variability. This multi-decadal variability of streamflows is generally weaker in hydrological simulations done with historical simulations from climate models. The climate projections have been downscaled with the method developed in this work. The temperature on the country, on average over climate models, could increased by 3,5°C in winter and 6,5°C in summer in the course of this century. Precipitations will decrease all over the country in summer, nearly by half on southern part of France for the most severe scenario. In winter, precipitations will increase in the northern part of the country and will decrease slightly in the southern part. In the next few decades, the decrease in precipitation is important in summer, and changes are less pronounced for other seasons. Results of hydrological projections done with one hydrological model and an ensemble of climate models are presented for the coming decades and for the end of the century. On the Seine river, results slightly differ in winter from those presented in previous studies. Here, precipitations and streamflow increase in winter and decrease in summer on that river basin. Elsewhere in France, results are consistent with previous studies, namely an increase in evapotranspiration, a decrease in streamflow and much drier soil. The uncertainty due to both climate models and internal variability on relative changes in streamflows always increase during the 21st century, to over 20% in winter for the most severe scenario. In the coming decades, the uncertainty due to internal variability only on streamflow changes is as strong as the uncertainty due to both climate models and internal variability. In the coming decades, annual streamflow changes of the Loire, Garonne and Rhône rivers are stronger than the maximum changes observed during the 20th century.L'étude des impacts du changement climatique demande souvent de mettre en place de longues chaînes de modélisation. Du modèle qui servira à estimer les concentrations futures en gaz à effet de serre jusqu'au modèle d'impact. Tout au long de cette chaîne de modélisation, les sources d'incertitudes s'accumulent et compliquent l'exploitation des résultats pour l'élaboration de stratégies d'adaptation. Il est proposé ici d'évaluer les impacts du changement climatique sur le cycle hydrologique en France ainsi que les incertitudes qui y sont associées. La contribution de chacune des sources d'incertitudes n'est pas abordée, principalement celle associée aux scénarios d'émission de gaz à effet de serre, aux modèles climatiques et à la variabilité interne. Nous proposons dans ce travail une approche pour évaluer la transférabilité dans un climat futur de la méthode statistique de régionalisation des simulations climatiques. La vérification de l'hypothèse de transférabilité effectuée est l'une des principales sources d'incertitudes des méthodes statistiques de régionalisation. L'évaluation proposée ici s'appuie sur l'utilisation de modèles régionaux, dans un cadre dit de modèle parfait, et permet de montrer que l'utilisation de certain prédicteurs s'avèrent utile à assurer la transférabilité de la méthode de régionalisation dans un climat futur. Cette approche proposée pour une méthode de désagrégation statistique est également applicable à des méthodes de correction des biais des modèles régionaux. Les récentes réanalyses atmosphériques sur l'ensemble du XXème siècle, régionalisées avec la méthode développée dans ce travail, et associées aux observations de température et précipitations permettent de caractériser le cycle hydrologique en France. Elles permettent notamment de montrer que la variabilité multi-décennale des débits observés pendant le XXème siècle est généralisée à l'ensemble du pays et est liée à la variabilité des conditions atmosphériques. Cette variabilité multi-décennale des débits est généralement plus faible dans les simulations hydrologiques réalisées avec les simulations historiques des modèles climatiques. Les projections climatiques ont été régionalisées avec la méthode développée dans ce travail. La température sur l'ensemble du pays, en moyenne sur les modèles climatiques, augmente jusqu'à 3,5°C en hiver et 6,5°C en été d'ici la fin du siècle. Les précipitations vont diminuer sur l'ensemble du pays en été, de presque moitié sur le sud du pays pour le scénario le plus sévère. En hiver, elles augmentent sur la moitié nord du pays et diminuent légèrement sur la partie sud. Dès les prochaines décennies, la diminution des précipitations est importante en été, l'évolution est moins marquée pour les autres saisons. Enfin, les résultats des projections hydrologiques réalisées avec un modèle hydrologique et un ensemble de modèles climatiques sont présentés pour les prochaines décennies et également pour la fin du XXIème siècle. Sur la Seine, les résultats sont différents en hiver de ceux présentés dans de précédentes études. Ici, les précipitations et les débits augmentent en hiver et diminuent en été sur ce bassin versant. Ailleurs en France, les résultats convergent avec les études précédentes, à savoir une augmentation de l'évapotranspiration, une diminution généralisée des débits et un assèchement des sols. L'incertitude due aux modèles climatiques et à la variabilité interne sur les changements relatifs de débits augmente systématiquement pendant le XXIème siècle, jusqu'à atteindre plus de 20% en hiver pour le scénario le plus sévère. Dans les prochaines décennies, l'incertitude due uniquement à la variabilité interne sur les changements de débits est aussi forte que l'incertitude due aux modèles climatiques et à la variabilité interne. Dès les prochaines décennies, les changements de débits annuels sont plus forts sur la Loire, la Garonne et le Rhône que les changements maximaux observés pendant le XXème siècle

Cerebrospinal Fluid Proteome Alterations Associated with Neuropsychiatric Symptoms in Cognitive Decline and Alzheimer's Disease

Although neuropsychiatric symptoms (NPS) are common and severely affect older people with cognitive decline, little is known about their underlying molecular mechanisms and relationships with Alzheimer's disease (AD). The aim of this study was to identify and characterize cerebrospinal fluid (CSF) proteome alterations related to NPS. In a longitudinally followed-up cohort of subjects with normal cognition and patients with cognitive impairment (MCI and mild dementia) from a memory clinic setting, we quantified a panel of 790 proteins in CSF using an untargeted shotgun proteomic workflow. Regression models and pathway enrichment analysis were used to investigate protein alterations related to NPS, and to explore relationships with AD pathology and cognitive decline at follow-up visits. Regression analysis selected 27 CSF proteins associated with NPS. These associations were independent of the presence of cerebral AD pathology (defined as CSF p-tau181/Aβ1-42 > 0.0779, center cutoff). Gene ontology enrichment showed abundance alterations of proteins related to cell adhesion, immune response, and lipid metabolism, among others, in relation to NPS. Out of the selected proteins, three were associated with accelerated cognitive decline at follow-up visits after controlling for possible confounders. Specific CSF proteome alterations underlying NPS may both represent pathophysiological processes independent from AD and accelerate clinical disease progression. Keywords: Alzheimer’s disease; cognitive decline; proteom

Quantitative analysis of protein glycation in clinical samples

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Motif affinity and mass spectrometry proteomic approach for the discovery of cellular AMPK targets: identification of mitochondrial fission factor as a new AMPK substrate

AMP-activated protein kinase (AMPK) is a key cellular energy sensor and regulator of metabolic homeostasis. Although it is best known for its effects on carbohydrate and lipid metabolism, AMPK is implicated in diverse cellular processes, including mitochondrial biogenesis, autophagy, and cell growth and proliferation. To further our understanding of energy homeostasis through AMPK-dependent processes, the design and application of approaches to identify and characterise novel AMPK substrates are invaluable. Here, we report an affinity proteomicstrategy for the discovery and validation of AMPK targets using an antibody to isolate proteins containing the phospho-AMPK substrate recognition motif from hepatocytes that had been treated with pharmacological AMPK activators. We identified 57 proteins that were uniquely enriched in the activator-treated hepatocytes, but were absent in hepatocytes lacking AMPK. We focused on two candidates, cingulin and mitochondrial fission factor (MFF), and further characterised/validated them as AMPK-dependent targets by immunoblotting with phosphorylation site-specific antibodies. A small-molecule AMPK activator caused transient phosphorylation of endogenous cingulin at S137 in intestinal Caco2 cells. Multiple splice-variants of MFF appear to express in hepatocytes and we identified a common AMPK-dependent phospho-site (S129) in all the 3 predominant variants spanning the mass range and a short variant-specific site (S146). Collectively, our proteomic-based approach using a phospho-AMPK substrate antibody in combination with genetic models and selective AMPK activators will provide a powerful and reliable platform for identifying novel AMPK-dependent cellular targets