Criblage d'inhibiteurs réversibles et irréversibles des phosphatases CDC25s par spectrométrie de masse - Application à des extraits d'origine végétale

The CDC25s phosphatases are key regulators of the physiological cell cycle progression. Their overexpression has been reported in a significant number of cancers and their inhibition appears to be an interesting strategy for treatments. We propose here a rapid screening test allowing the detection of reversible and irreversible CDC25 inhibitors. The test is based on the incubation of the candidate molecules with the human CDC25 proteins followed by an ultrafiltration step. The retentate is then directly analyzed by MALDI-TOFMS to detect reversible inhibitors or submitted to PMF analysis to reveal irreversible inhibitors. In parallel we applied this test to vegetable extracts in order to identify novel CDC25 inhibitors. The CDC25s inhibitory activity of these extracts is also evaluated in vitro thanks to biological tests. It results from this work that one molecule never known for its activity on the CDC25s shows a potential inhibitory effect and is cytotoxic on human cellular lineageLes protéines CDC25 (Cell Cycle Division 25) sont des régulateurs clés de la progression du cycle cellulaire et leur surexpression a été reportée dans de nombreux types de cancers. Leur inhibition apparait donc intéressante dans le cadre de traitements anticancéreux. L'objet de ces recherches a été de développer un test de criblage permettant de détecter les inhibiteurs réversibles et irréversibles des CDC25s, puis d'appliquer ce test à des extraits végétaux. La première partie implique la spectrométrie de masse par désorption/ionisation laser assistée par matrice couplée à un détecteur à temps de vol (MALDI-TOFMS) afin de détecter les molécules se liant aux CDC25s avec une liaison faible (inhibiteurs réversibles). La deuxième partie de ce test beaucoup plus innovante consiste à mettre en évidence les ligands se liant avec une liaison plus forte (inhibiteurs irréversibles) aux CDC25s. Pour cela, une étape de digestion trypsique des protéines est réalisée, puis l'étude de l'empreinte peptidique massique est procédée sur le digest afin de déceler les inhibiteurs irréversibles. En parallèle, nous nous sommes intéressés à l'application de ce test à des extraits végétaux dans le but d'identifier de nouveaux inhibiteurs des CDC25s. L'activité inhibitrice de CDC25s de ces extraits est également évaluée in vitro grâce à des tests biologiques visant à compléter les premiers résultats obtenus lors du criblage par spectrométrie de masse. De ces travaux il résulte qu'une molécule encore non connue pour son activité sur CDC25s a montré un potentiel effet inhibiteur de ces protéines et cytotoxique sur lignées cellulaires humaine

Screening of reversible and irreversible inhibitors of CDC25 phosphatases by mass spectrometry : Application to vegetable extracts

Les protéines CDC25 (Cell Cycle Division 25) sont des régulateurs clés de la progression du cycle cellulaire et leur surexpression a été reportée dans de nombreux types de cancers. Leur inhibition apparait donc intéressante dans le cadre de traitements anticancéreux. L'objet de ces recherches a été de développer un test de criblage permettant de détecter les inhibiteurs réversibles et irréversibles des CDC25s, puis d'appliquer ce test à des extraits végétaux. La première partie implique la spectrométrie de masse par désorption/ionisation laser assistée par matrice couplée à un détecteur à temps de vol (MALDI-TOFMS) afin de détecter les molécules se liant aux CDC25s avec une liaison faible (inhibiteurs réversibles). La deuxième partie de ce test beaucoup plus innovante consiste à mettre en évidence les ligands se liant avec une liaison plus forte (inhibiteurs irréversibles) aux CDC25s. Pour cela, une étape de digestion trypsique des protéines est réalisée, puis l'étude de l'empreinte peptidique massique est procédée sur le digest afin de déceler les inhibiteurs irréversibles. En parallèle, nous nous sommes intéressés à l'application de ce test à des extraits végétaux dans le but d'identifier de nouveaux inhibiteurs des CDC25s. L'activité inhibitrice de CDC25s de ces extraits est également évaluée in vitro grâce à des tests biologiques visant à compléter les premiers résultats obtenus lors du criblage par spectrométrie de masse. De ces travaux il résulte qu'une molécule encore non connue pour son activité sur CDC25s a montré un potentiel effet inhibiteur de ces protéines et cytotoxique sur lignées cellulaires humainesThe CDC25s phosphatases are key regulators of the physiological cell cycle progression. Their overexpression has been reported in a significant number of cancers and their inhibition appears to be an interesting strategy for treatments. We propose here a rapid screening test allowing the detection of reversible and irreversible CDC25 inhibitors. The test is based on the incubation of the candidate molecules with the human CDC25 proteins followed by an ultrafiltration step. The retentate is then directly analyzed by MALDI-TOFMS to detect reversible inhibitors or submitted to PMF analysis to reveal irreversible inhibitors. In parallel we applied this test to vegetable extracts in order to identify novel CDC25 inhibitors. The CDC25s inhibitory activity of these extracts is also evaluated in vitro thanks to biological tests. It results from this work that one molecule never known for its activity on the CDC25s shows a potential inhibitory effect and is cytotoxic on human cellular lineag

Criblage d'inhibiteurs réversibles et irréversibles des phosphatases CDC25s par spectrométrie de masse (Application à des extraits d'origine végétale)

Les protéines CDC25 (Cell Cycle Division 25) sont des régulateurs clés de la progression du cycle cellulaire et leur surexpression a été reportée dans de nombreux types de cancers. Leur inhibition apparait donc intéressante dans le cadre de traitements anticancéreux. L'objet de ces recherches a été de développer un test de criblage permettant de détecter les inhibiteurs réversibles et irréversibles des CDC25s, puis d'appliquer ce test à des extraits végétaux. La première partie implique la spectrométrie de masse par désorption/ionisation laser assistée par matrice couplée à un détecteur à temps de vol (MALDI-TOFMS) afin de détecter les molécules se liant aux CDC25s avec une liaison faible (inhibiteurs réversibles). La deuxième partie de ce test beaucoup plus innovante consiste à mettre en évidence les ligands se liant avec une liaison plus forte (inhibiteurs irréversibles) aux CDC25s. Pour cela, une étape de digestion trypsique des protéines est réalisée, puis l'étude de l'empreinte peptidique massique est procédée sur le digest afin de déceler les inhibiteurs irréversibles. En parallèle, nous nous sommes intéressés à l'application de ce test à des extraits végétaux dans le but d'identifier de nouveaux inhibiteurs des CDC25s. L'activité inhibitrice de CDC25s de ces extraits est également évaluée in vitro grâce à des tests biologiques visant à compléter les premiers résultats obtenus lors du criblage par spectrométrie de masse. De ces travaux il résulte qu'une molécule encore non connue pour son activité sur CDC25s a montré un potentiel effet inhibiteur de ces protéines et cytotoxique sur lignées cellulaires humainesThe CDC25s phosphatases are key regulators of the physiological cell cycle progression. Their overexpression has been reported in a significant number of cancers and their inhibition appears to be an interesting strategy for treatments. We propose here a rapid screening test allowing the detection of reversible and irreversible CDC25 inhibitors. The test is based on the incubation of the candidate molecules with the human CDC25 proteins followed by an ultrafiltration step. The retentate is then directly analyzed by MALDI-TOFMS to detect reversible inhibitors or submitted to PMF analysis to reveal irreversible inhibitors. In parallel we applied this test to vegetable extracts in order to identify novel CDC25 inhibitors. The CDC25s inhibitory activity of these extracts is also evaluated in vitro thanks to biological tests. It results from this work that one molecule never known for its activity on the CDC25s shows a potential inhibitory effect and is cytotoxic on human cellular lineageNANCY-INPL-Bib. électronique (545479901) / SudocSudocFranceF

Sample preparation and analysis of gangliosides and phospholipids from plasma by liquid chromatography coupled to mass spectrometry

Gangliosides (GG) and phospholipids (PL) are two classes of complex and polar lipids exhibiting a wide structural heterogeneity. LC/MS offers an access to this huge diversity but also faces some challenges. Efficient extraction and purification sample methods are thus required to perform accurate and reliable analyses. To this end, GG and PL were extracted from human plasma with three solvent mixtures (CHCl3/MeOH/H2O) of increasing polarity, in comparison with the reference Folch method. GG were further purified with a Phree Phospholipid Removal column, whose sorbent is designed to selectively remove PL. GG and PL classes were then separated under HILIC conditions before analysis using a QqQ mass spectrometer.Our method enabled a fast, straightforward and efficient extraction of both complex lipids, PL and GG. The extraction efficiency was much higher than the Folch method for the especially polar lipids that are GG and Lyso-PC, and similar for the various PL classes (PG, PI, PE, PS, PC, Sphingomyelin). The relative quantitative analysis of GG, performed by single reaction monitoring thanks to their characteristic sialic acid fragment (m/z 290), revealed a massive ion-suppression effect (up to 90%) due to co-elution of PC with the less polar GG. Removal of PL is thus essential for an accurate GG analysis by LC/MS and the Phree Phospholipid Removal column appeared as a powerful tool to achieve this purification step.Our sample preparation method allows the accurate analysis of both GG and PL classes from a single biological extract

Gangliosides characterization by high resolution mass spectrometry and ion mobility

Gangliosides (GG) are sialic acid-containing glycosphingolipids particularly abundant in the nervous system. They exhibit a wide variety of structures depending on both the oligosaccharide chain and the ceramide moiety. About 200 GG species have been described so far, but their diversity and biological roles are far from being completely elucidated. An efficient method of identification and quantification is crucial to apprehend this huge heterogeneity. In this study, we focused on the characterization of the ceramide portion of GG, identifying the long chain base (LCB) and the fatty acid. A commercial standard of GD3 was analysed with both a Thermo LTQ-Orbitrap XLTM and a Waters Synapt G2-Si equipped with ion mobility.Whatever the equipment used, fragmentation of GD3 40:1 molecular species revealed the presence of four LCBs (d16:1, d17:1, d17:0 and d18:1) for one ceramide type, suggesting the presence of four GG isomers. This observation was confirmed by the MS3 analyses conducted with the Orbitrap, which allowed isolation and fragmentation of the ion corresponding to the ceramide. The technique of ion mobility offered the possibility to separate the four detected LCBs, after MS/MS fragmentation of GD3 40:1. We were thus able to detect and separate the LCBs, according to their differences of steric hindrance, for each isomer of GG. In this way we deduced the combination LCB/fatty acid (ex: GD3 d16:1/24:0) for each GD3 molecular species. Moreover, this separation presents the advantage to individualize each molecular isobaric species of GG for their ulterior quantification.Ion mobility applied to GG analysis allowed separation and further characterization and quantification of the various ceramide isomers

Rapid sample preparation for ganglioside analysis by liquid chromatography mass spectrometry

Indexation en cours.International audienceGangliosides (GG) are glycosphingolipids, composed of a ceramide moiety (fatty acid and long chain base) linked to an oligosaccharide chain containing one (or more) molecule of sialic acid. After lipid extraction from biological matrices, quantification of GG by liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI/MS) can be impacted by ion suppression effects due to co-elution with more abundant lipids in the matrix. In this study, a simple, rapid and efficient method to purify GG from biological samples by Phree columns is proposed. This approach proved to be useful in eliminating phospholipids (PL) from the matrix and thus increasing the signal of GG classes and molecular species in rat brain samples during LC-ESI/MS analysis

Biological activity of Sapindus mukorossi Gaerten (Sapindaceae) aqueous extract against Thysanoplusia orichalcea (Lepidoptera: Noctuidae)

International audienceSpearmint is one of the crops especially threatened by Thysanoplusia orichalcea. For controlling pests, producers use synthetic insecticides exposing consumers to intoxication risks. To seek for alternative products to synthetic insecticides, aqueous extracts from Sapindus mukorossi pericarp were tested against T. orichalcea larvae. The dry extract represented 48.5% of the dry weight of S. mukorossi pericarp and it contains 75.3% of saponins. The chemical composition in saponin was checked by mass spectrometry. Aqueous solutions containing this extract at four different concentrations were applied topically on eggs or ingested by larvae of T. orichalcea to evaluate mortality, leaf consummation, development duration and weight of larvae of the insect. Topical application of pericarp aqueous extract of S. mukorossi fruit had no effects on eggs or larvae of T. orichalcea. By ingestion, the tested extract significantly affected survivals, consummation, growth and development of larvae. In regards to control, the larval consummation was reduced between 40 and 100%, according to concentration, exposure duration and stage used. The extract delayed larval development by I to 2 days. The larval weights were reduced by 7 to 68%. The use of aqueous extract of pericarp fruit of S. mukorossi is discussed in terms of integrated pest management program concerning T. orichalcea on spearmint crop

Apprehending ganglioside diversity: a comprehensive methodological approach

Gangliosides make a wide family of glycosphingolipids ubiquitously expressed in mammalian tissues and particularly abundant in the brain and nervous system. They exhibit a huge diversity due to structural variations in both their oligosaccharidic chain and ceramide moiety, which represent a real analytical challenge. Since their discovery in the 1940s, methods have persistently improved until the emergence of Liquid Chromatography/Mass Spectrometry (LC/MS) which offers a high level of specificity and sensitivity and is suitable with high-throughput profiling studies. We describe here a comprehensive approach relying on various techniques and aiming at fully characterizing gangliosides in biological samples. First, total ganglioside content was determined by a biochemical assay. Second, ganglioside class composition was assessed by high performance thin layer chromatography followed by colorimetric revelation. Then, ceramide types of ganglioside classes were identified and their relative quantification was performed thanks to the development of a powerful and reliable LC/MS method. Finally, ceramides were structurally characterized and minor and less common ganglioside classes were identified using high resolution MS. These methods were applied to the rat retina to provide an exhaustive description of its ganglioside composition, giving the base for a better understanding of the precise roles of gangliosides in this tissue