MALDI/MS peptide mass fingerprinting for proteome analysis: identification of hydrophobic proteins attached to eucaryote keratinocyte cytoplasmic membrane using different matrices in concert

BACKGROUND: MALDI-TOF-MS has become an important analytical tool in the identification of proteins and evaluation of their role in biological processes. A typical protocol consists of sample purification, separation of proteins by 2D-PAGE, enzymatic digestion and identification of proteins by peptide mass fingerprint. Unfortunately, this approach is not appropriate for the identification of membrane or low or high pI proteins. An alternative technique uses 1D-PAGE, which results in a mixture of proteins in each gel band. The direct analysis of the proteolytic digestion of this mixture is often problematic because of poor peptide detection and consequent poor sequence coverage in databases. Sequence coverage can be improved through the combination of several matrices. RESULTS: The aim of this study was to trust the MALDI analysis of complex biological samples, in order to identify proteins that interact with the membrane network of keratinocytes. Peptides obtained from protein trypsin digestions may have either hydrophobic or hydrophilic sections, in which case, the direct analysis of such a mixture by MALDI does not allow desorbing of all peptides. In this work, MALDI/MS experiments were thus performed using four different matrices in concert. The data were analysed with three algorithms in order to test each of them. We observed that the use of at least two matrices in concert leads to a twofold increase of the coverage of each protein. Considering data obtained in this study, we recommend the use of HCCA in concert with the SA matrix in order to obtain a good coverage of hydrophilic proteins, and DHB in concert with the SA matrix to obtain a good coverage of hydrophobic proteins. CONCLUSION: In this work, experiments were performed directly on complex biological samples, in order to see systematic comparison between different matrices for real-life samples and to show a correlation that will be applicable to similar studies. When 1D gel is needed, each band may contain a great number of proteins, each present in small amounts. To improve the proteins coverage, we have performed experiments with some matrices in concert. These experiments enabled reliable identification of proteins, without the use of Nanospray MS/MS experiments

International Expert Consensus on Switching Platelet P2Y(12) Receptor-Inhibiting Therapies

Dual antiplatelet therapy with aspirin and a P2Y(12) inhibitor is the treatment of choice for the prevention of atherothrombotic events in patients with acute coronary syndromes and for those undergoing percutaneous coronary interventions. The availability of different oral P2Y(12) inhibitors (clopidogrel, prasugrel, ticagrelor) has enabled physicians to contemplate switching among therapies because of specific clinical scenarios. The recent introduction of an intravenous P2Y(12) inhibitor (cangrelor) further adds to the multitude of modalities and settings in which switching therapies may occur. In clinical practice, it is not uncommon to switch P2Y(12) inhibitor, and switching may be attributed to a variety of factors. However, concerns about the safety of switching between these agents have emerged. Practice guidelines have not fully elaborated on how to switch therapies, leaving clinicians with limited guidance on when and how to switch therapies when needed. This prompted the development of this expert consensus document by key leaders from North America and Europe with expertise in basic, translational, and clinical sciences in the field of antiplatelet therapy. This expert consensus provides an overview of the pharmacology of P2Y(12) inhibitors, different modalities and definitions of switching, and available literature and recommendations for switching between P2Y(12) inhibitors

Etude des proprietes fonctionnelles de l'endopeptidase neutre cerebrale

SIGLECNRS TD Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Identification de marqueurs de prolifération et de différenciation des kératinocytes de l'épiderme humain

Les kératinocytes souches de la couche basale de l'épiderme humain assurent l'auto-renouvellement de ce tissu. Lors de leur différenciation, les kératinocytes migrent vers les couches supérieures de l'épiderme jusqu'à la desquamation. Les mécanismes de différenciation des kératinocytes en culture sont mal connus. L'apparition de nouvelles technologies comme les puces à ADN ou l'analyse protéomique nous a permis d'identifier des gènes et des protéines exprimés au cours de la différenciation des kératinocytes. Dans le but de mieux caractériser les cellules souches, nous avons entrepris l'identification de protéines membranaires des kératinocytes de la couche basale. En isolant des microdomaines membranaires de ces cellules, nous avons pu identifier par analyse protéomique des protéines représentant des marqueurs potentiels des kératinocytes de la couche basale. Cette nouvelle approche a permis d'isoler des kératinocytes de la couche basale et par conséquent, les cellules souches.Keratinocyte stem cells of the basal layer of the epidermis ensure the self-renewal of this tissue. Their progeny differentiate as they migrate towards the squamous layer. The mechanisms of human keratinocyte differentiation are still poorly known. The emergence of new technologies, such as cDNA microarrays or proteomic analysis, allowed us to identify, in tissue culture, genes and proteins expressed in proliferating versus differentiating keratinocytes. In order to further characterize keratinocyte stem cells, the identification of plasma membrane markers of basal keratinocytes was performed by isolating membrane microdomains from keratinocytes. By proteomic approach, we identified proteins representing potential cell surface markers of the epidermis. Altogether, these results allowed us to define a new strategy to isolate progenitors of basal cells and consequently keratinocyte stem cells.EVRY-BU (912282101) / SudocSudocFranceF

Inégalités sociales d'accès aux soins primaires (l'exemple des consultations inappropriées aux urgences)

PARIS6-Bibl. St Antoine CHU (751122104) / SudocSudocFranceF

Analyse fonctionnelle des mutations du gène GJB6 codant la connexine30 humaine responsable du syndrome de Clouston

La dysplasie ectodermique hidrotique ou syndrome de Clouston est une génodermatose rare qui se caractérise par une hyperkératose palmoplantaire, une alopécie généralisée et une dystrophie des ongles. Nous avons démontré que ce syndrome est dû à des mutations dans le gène GJB6, qui code la connexine30 humaine, pour lequel nous avons déterminé la structure génomique et analysé la région promotrice. L'analyse fonctionnelle des formes mutées de la protéine a permis de démontrer que leur transport bénéficie de la présence de la Cx30 sauvage et que les jonctions communicantes formées de Cx30 mutées sont fonctionnelles. Cependant, les Cx30 mutées se sont avérées capables de former des hemicanaux non-appariés constitutivement ouverts à la surface de la cellule qui entraînent une fuite d'ATP vers le milieu extracellulaire. Cette observation nous a permis d'émettre l'hypothèse que cette libération d'ATP est à l'origine du phénotype observé au niveau de l'épiderme atteint.EVRY-BU (912282101) / SudocSudocFranceF