Surface Plasmon Resonance coupled to Mass Spectrometry to studylectin-sugar interactions

International audienceBiomolecular interactions are at the heart of the functioning of all living systems. The study ofbiointeractions is essential for understanding the global organization of the cellular machinery and theirrole in physiological processes. They constitute a significant challenge in analytical chemistry,diagnostics, and therapeutic research.Lectins are proteins that specifically recognize edible sugars. Our project aims to develop a couplingbetween Surface Plasmon Resonance Imaging and Mass Spectrometry (SPRi -MS) to analyze protein-sugar interactions. The aim is to create a multiplex SPR biochip with immobilized lectins and then to usethis biochip in coupling with MALDI-TOF-MS. This coupling allows the kinetics and thermodynamics ofthe interaction to be studied in real-time, together with the structural identification of the sugarscaptured from a complex mixture.By SPRi, this work confirmed significant interactions, between the lectin WGA and the neoglycosylatedBSA gratied carrying the sugars N-acetylgalactosamine and N-acetylglucosamine, and between the lectinAIA and neoglycosylated BSA carrying galactose and its N-acetylated form. We atempted the MSdetection of captured glycosylated BSA directly from the biochip surface; however, the lack of sensitivityin MS detection hindered the development of the coupling. The sensitivity depends, on the amount ofligands retained on biochip surface, which itself depends, among other factors, on the chemicalfunctionalization of the biochip surface, the nature of the receptors and their immobilization on thesurface [1]. Modifications of the MALDI-TOF-MS analysis conditions are being carried out with the useof MALDI imaging and the use of alternative receptors in order to evaluate their impact on the sensitivityof detection by SPRi-MS

Coupling of Surface Plasmon Resonance with Mass Spectrometry to study protein glycosylation

International audienceLectins are widely appreciated, in the fields of hematology and immunology, due to their specificbinding to carbohydrate structures. Lectins are commonly present on the cell surface or elsewhere; they arealso known as specific markers for membrane glycoprotein structures. This study is focused on the use ofJacalin lectin (Artocarpus integrifolia) (AIA), which belongs to the family of galactose-binding lectins and isused here as a model of lectin. The lack of sensitivity in MS detection hinders the development of approachesusing Surface Plasmon Resonance (SPR) coupled with Mass Spectrometry (MS). Among the factors affectingsensitivity, the amount of ligands retained on the biochip surface is crucial [1]. Thanks to their smaller size andmultimerism, lectins offer an attractive receptor alternative to usual antibodies for increasing ligand capture,which is particularly interesting in glycoconjugates analysis.Our project aims to develop a multiplex SPR biochip with immobilized lectins to afford the couplingbetween SPR Imaging and MS (SPRi-MS) to determine protein glycosylation and further study of unknownlectins and their interactions with N-glycans. This coupling allows the real-time monitoring of interactions andthe determination of their kinetics and thermodynamics parameters, together with the structuralidentification of the glycoconjugates captured from a complex mixture [1], [2], [3].Fetuin is a plasma glycoprotein containing N-acetylglucosamine (GlcNac) and N-acetylgalactosamine(GalNac) in its structure, which is commonly used as a model for N- and O-glycans analysis. Our SPRi analysisconfirmed significant interactions between AIA and fetuin. The sensorgrams showed considerable variationsin reflectivity accompanied by the lighting of the spots upon the injection of increasing concentrations offetuin. Detection of the captured fetuin was successfully achieved by MALDI-TOF MS directly on the lectinbiochip.Moreover, a protocol for the on-chip spraying of the MALDI matrix is currently in development in thelaboratory. This mode of matrix deposit will be combined with a direct on-chip MALDI-TOF MS imaging (MSi)experiment to evaluate the impact on both the sensitivity and the selectivity of detection of fetuin by a SPRi-MSi approach

Rationale, design, and baseline characteristics in Evaluation of LIXisenatide in Acute Coronary Syndrome, a long-term cardiovascular end point trial of lixisenatide versus placebo

BACKGROUND: Cardiovascular (CV) disease is the leading cause of morbidity and mortality in patients with type 2 diabetes mellitus (T2DM). Furthermore, patients with T2DM and acute coronary syndrome (ACS) have a particularly high risk of CV events. The glucagon-like peptide 1 receptor agonist, lixisenatide, improves glycemia, but its effects on CV events have not been thoroughly evaluated. METHODS: ELIXA (www.clinicaltrials.gov no. NCT01147250) is a randomized, double-blind, placebo-controlled, parallel-group, multicenter study of lixisenatide in patients with T2DM and a recent ACS event. The primary aim is to evaluate the effects of lixisenatide on CV morbidity and mortality in a population at high CV risk. The primary efficacy end point is a composite of time to CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for unstable angina. Data are systematically collected for safety outcomes, including hypoglycemia, pancreatitis, and malignancy. RESULTS: Enrollment began in July 2010 and ended in August 2013; 6,068 patients from 49 countries were randomized. Of these, 69% are men and 75% are white; at baseline, the mean ± SD age was 60.3 ± 9.7 years, body mass index was 30.2 ± 5.7 kg/m(2), and duration of T2DM was 9.3 ± 8.2 years. The qualifying ACS was a myocardial infarction in 83% and unstable angina in 17%. The study will continue until the positive adjudication of the protocol-specified number of primary CV events. CONCLUSION: ELIXA will be the first trial to report the safety and efficacy of a glucagon-like peptide 1 receptor agonist in people with T2DM and high CV event risk