Immuno-MALDI-MS in Human Plasma and on-Chip Biomarker Characterizations at the Femtomole Level

Immuno-SPR-MS is the combination of immuno-sensors in biochip format with mass spectrometry. This association of instrumentation allows the detection and the quantification of proteins of interest by SPR and their molecular characterization by additional MS analysis. However, two major bottlenecks must be overcome for a wide diffusion of the SPR-MS analytical platform: (i) To warrant all the potentialities of MS, an enzymatic digestion step must be developed taking into account the spot formats on the biochip and (ii) the biological relevancy of such an analytical solution requires that biosensing must be performed in complex media. In this study, we developed a procedure for the detection …

Human infant saliva peptidome is modified with age and diet transition.

Abstract In order to describe developmental changes in human salivary peptidome, whole saliva was obtained from 98 infants followed longitudinally at 3 and 6 months of age. Data on teeth eruption and diet at the age of 6 months were also recorded. Salivary peptide extracts were characterised by label-free MALDI-MS. Peptides differentially expressed between the two ages, and those significantly affected by teeth eruption or introduction of solid foods were identified by MALDI TOF–TOF and LC ESI MS–MS. Out of 81 peaks retained for statistical analysis, 26 were overexpressed at the age of 6 months. Exposure to solid foods had a more pronounced effect on profiles (overexpression of nine peaks) …

Identification of HSP90 as a new GABARAPL1 (GEC1)-interacting protein

GABARAPL1 belongs to the small family of GABARAP proteins (including GABARAP, GABARAPL1 and GABARAPL2/GATE-16), one of the two subfamilies of the yeast Atg8 orthologue. GABARAPL1 is involved in the intracellular transport of receptors, via an interaction with tubulin and GABA(A) or kappa opioid receptors, and also participates in autophagy and cell proliferation. In the present study, we identify the HSP90 protein as a novel interaction partner for GABARAPL1 using GST pull-down, mass spectrometry and coimmunoprecipitation experiments. GABARAPL1 and HSP90 partially colocalize in MCF-7 breast cancer cells overexpressed Dsred-GABARAPL1 and in rat brain. Moreover, treatment of MCF-7 cells overe…

Associations between food consumption patterns and saliva composition: Specificities of eating difficulties children

Identifying objective markers of dietwould be beneficial to research fields such as nutritional epidemiology. As a preliminary study on the validity of using saliva for this purpose, and in order to explore the relationship between saliva and diet, we focused on clearly contrasted groups of children: children with eating difficulties (ED) receiving at least 50% of their energy intake through artificial nutrition vs healthy controls (C). Saliva of ED and C children was analyzed by various methods (targeted biochemical analyses, 2-D electrophoresis coupled to MS, 1H NMR) and their diet was characterized using food frequency questionnaires, considering 148 food items grouped into 13 categories…

Flavour and Mass Spectrometry

The flavour of food, commonly called ‘taste’, is an essential determinant of food choice by the consumer. Flavour perception in the mouth results from at least two sensory modalities, aroma and taste, due to odour-active and taste-active compounds released from the food matrix on eating. Instrumental assessment of flavour should characterise odouriferous molecules present in the volatile organic compounds of food and sapid components, essentially present in the non-volatile fraction of food. Mass spectrometry (MS) plays a fundamental role in the studies dedicated to flavour characterisation. Coupled with gas chromatography (GC) and benefitting from the latest development in multidimensional…

The production of an infant formula with a minimally processed route impacts its nutritional, physiological and sensorial qualities

International audience

Technological innovation around protein and cell biochip for diagnosis: a translational research from nanoworld to patient

International audience; A great challenge in biosensors and diagnosis devices relies on the way to reconstitute relevant biological mechanisms on surface of the biochips and which analytical tools are convenient to provide accurate and rapid information on the structures and function of molecules attached to this surface. A better control in the realization of biochips can be obtained in combining different complementary approaches while always keeping in mind the biological key point. Researches in CLIPP are focused towards this objective. Conception, realization and characterization of protein and cell chips are presented. We detail different strategies of materials engineering1,2,3, chem…

Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells

IF 5.547; International audience; Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence bo…

Characterization and purification of a bacterial chlorogenic acid esterase detected during the extraction of chlorogenic acid from arbuscular mycorrhizal tomato roots

International audience; A Gram-negative bacterium able to grow using chlorogenic acid (5-caffeoylquinic acid) as sole carbon source has been isolated from the roots of tomato plants inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis. An intracellular esterase exhibiting very high affinity (K-m = 2 mu M) for chlorogenic acid has been extracted and purified by FPLC from the chlorogenate-grown cultures of this bacterium. The molecular mass of the purified esterase determined by SDS-PAGE was 61 kDa and its isoelectric point determined by chromatofocusing was 7.75. The esterase hydrolysed chlorogenic acid analogues (caffeoylshikimate, and the 4- and 3-caffeoylquinic acid i…

Toward the Identification of Two Glycoproteins Involved in the Stomatal Deregulation of Downy Mildew–Infected Grapevine Leaves

SPE Pôle IPM UB; International audience; Stomata remain abnormally opened and unresponsive to abscisic acid in grapevine leaves infected by downy mildew. This deregulation occurs from 3 days post inoculation and increases concomitantly with leaf colonization by the pathogen. Using epidermal peels, we demonstrated that the active compound involved in this deregulation is located in the apoplast. Biochemical assays showed that the active compound present in the apoplastic fluids isolated from Plasmopara viticola infected grapevine leaves (IAF) is a CysCys bridge-independent, thermostable and glycosylated protein. Fractionation guided assays based on chromatography / stomatal response and prot…

Glycated albumin with loss of fatty acid binding capacity contributes to enhanced arachidonate oxygenation and platelet hyperactivity: relevance in patients with type 2 diabetes.

High plasma concentrations of nonesterified fatty acids (NEFAs), transported bound to serum albumin, are associated with type 2 diabetes (T2D). The effects of albumin on platelet function were investigated in vitro. Modifications of albumin, such as those due to glycoxidation, were found in patients with T2D, and the consequences of these modifications on biological mechanisms related to NEFA handling were investigated. Mass spectrometry profiles of albumin from patients with T2D differed from those from healthy control subjects. Diabetic albumin showed impaired NEFA binding capacity, and both structural and functional alterations could be reproduced in vitro by incubating native albumin wi…

Proteomic analysis of sub-retinal deposits in age-related macular degeneration

National audience

Sensitivity to bitterness: may in-mouth proteolysis be involved?

Sensitivity to bitterness: may in-mouth proteolysis be involved?. 9. Pangborn sensory science symposium

Identification clinique par spectrométrie de masse MALDI-TOF des champignons filamenteux : construction de banques de données pour les dermatophytes et les Fusarium

Symplekin, a polyadenylation factor, prevents MOZ and MLL activity on HOXA9 in hematopoietic cells

International audience; MOZ and MLL encoding a histone acetyltransferase and a histone methyltransferase, respectively, are targets for recurrent chromosomal translocations found in acute myeloblastic or lymphoblastic leukemia. We have previously shown that MOZ and MLL cooperate to activate HOXA9 gene expression in hematopoietic stem/progenitors cells. To dissect the mechanism of action of this complex, we decided to identify new proteins interacting with MOZ. We found that the scaffold protein Symplekin that supports the assembly of polyadenylation machinery was identified by mass spectrometry. Symplekin interacts and co-localizes with both MOZ and MLL in immature hematopoietic cells. Its …