Investigation of deprotonation reactions on globular and denatured proteins at atmospheric pressure by ESSI-MS

Deprotonation reactions of multiply charged protein ions have been studied by introducing volatile reference bases at atmospheric pressure between an electrosonic spray ionization (ESSI) source and the inlet of a mass spectrometer. Apparent gas-phase basicities (GBapp) of different charge states of protein ions were determined by a bracketing approach. The results obtained depend on the conformation of the protein ions in the gas phase, which is linked to the type of buffer used (denaturing or nondenaturing). In nondenaturing buffer, the GBapp values are consistent with values predicted by the group of Kebarle using an electrostatic model (J. Mass Spectrom. 2002, 38, 618) based on the crystal structures, but taking into account salt bridges between ionized basic and acidic sites on the protein surface. A new basicity order for the most basic sites was therefore obtained. An excellent agreement with the charge residue model (CRM) is obtained when comparing the observed and calculated maximum charge state. Decharging of the proteins in the electrosonic spray process could be also useful in the study on noncovalent complexes, by decreasing repulsive electrostatic interactions. A unified mechanism of the ESSI process is propose

Exploring Deprotonation Reactions on Peptides and Proteins at Atmospheric Pressure by Electro-Sonic Spray Ionization-Mass Spectrometry (ESSI-MS)

Electrospray ionization mass spectrometry (ESI-MS) rapidly became established as the method of choice for the production of large biomolecular ions in the gas phase. For peptides and proteins, ESI leads to the formation of multiply charged ions, in both the positive and negative ion mode. The charge-state distribution is directly related to the conformation of the macromolecular ions and to proton transfer reactions in the gas phase. Deprotonation reactions of multiply charged peptide and protein ions were studied by introducing volatile reference bases at atmospheric pressure between an electro-sonic spray ionization (ESSI) source and the inlet of a mass spectrometer. This new set-up offers the unique possibility to measure the apparent gas-phase basicity GBapp of multiply charged ions by a bracketing approach. The proof of principle was made using bradykinin derivatives, substance P and insulin chain B. We obtained values in excellent agreement with known GBapp values obtained at low pressure. These experiments were extended to seven model proteins showing that the thermodynamical properties of protein ions are directly correlated to the amino-acid sequence and the conformation in the gas phase. We also demonstrated that salt bridges between ionized basic and acidic sites still exist in the gas phase, confirming that electro-sonic spray is a very soft ionization technique

Apport de l’élastographie par imagerie des ondes de cisaillement pour l’évaluation de la photo-polymerisation du collagène cornéen

Corneal collagen cross-linking (CXL) is a kind of minimaly invasive corneoplasty mainlybased on a biomechanical concept, which is very difficult to measure physically, and whichthe therapeutic efficacy understanding is complex.Principles, different protocols and resultsare summarized in this thesis in order to illustrate the usefulness of the experimental modelchosen in our experimentations about elastographic corneal shear wave imaging.The pathway of our experimental work have led to the choice of trans-epithelial CXL (TCXL)assisted by iontophoresis (I-CXL), performed in vivo, on rabbits eyes. Elastographicmeasurements we obtained after animals euthanasia have shown a significant change of thecorneal elasticity profile after CXL, successively tested in a dynamic and in a static fashion.Our results do confirm the biomechanical efficacy of the I-CXL procedure and give a moreprecise idea of the sole photo-polymerization effect by avoiding any confounding healingconcern. Technological issues for corneal elastography with shear wave imaging have beenraised in this thesis to develop a realistic strategy for the launch of a clinically useful device.Le cross-linking du collagène cornéen (CXL) est une cornéoplastie mini-invasive reposant surun concept biomécanique difficile à objectiver physiquement et dont les preuves del’efficacité thérapeutique sont d’interprétation complexe. Les principes, les nuances et lesrésultats du CXL sont colligés dans cette thèse afin de valider l’intérêt du modèleexpérimental choisi pour tester la pertinence de notre travail de recherche sur l’élastographiecornéenne par ondes de cisaillement.Notre cheminement expérimental a abouti au choix du modèle de CXL trans-épithélial (TCXL)assisté par iontophorèse (I-CXL), réalisé in vivo, sur oeil de lapin. Les mesuresélastographiques obtenues après euthanasie ont ainsi pu démontrer une modificationsignificative du profil d’élasticité de la cornée après CXL, testé successivement de manièredynamique et statique.Nos résultats confirment donc l’efficacité biomécanique instantanée du I-CXL et donnent uneidée plus précise de la valeur de la photo-polymérisation du tissu cornéen isolée desphénomènes liés à la cicatrisation. Les enjeux technologiques de l’élastographe cornéen paranalyse des ondes de cisaillement ont pu être définis afin de développer une stratégie de miseen oeuvre d’un système pertinent pour la pratique clinique

Which electrospray-based ionization method best reflects protein-ligand interactions found in solution? A comparison of ESI, nanoESI, and ESSI for the determination of dissociation constants with mass spectrometry

We present a comparison of three different electrospray-based ionization techniques for the investigation of noncovalent complexes with mass spectrometry. The features and characteristics of standard electrospray ionization (ESI), chip-based nanoESI, and electrosonic spray ionization (ESSI) mounted onto a hybrid quadrupole time-of-flight mass spectrometer were compared in their performance to determine the dissociation constant (K D) of the model system hen egg white lysozyme (HEWL) binding to N,N′,N″-triacetylchitotriose (NAG3). The best K D value compared with solution data were found for ESSI, 19.4 ± 3.6 µM. Then, we determined the K Ds of the two nucleotide binding sites of adenylate kinase (AK), where we obtained K Ds of 2.2 ± 0.8 µM for the first and 19.5 ± 8.0 µM for the second binding site using ESSI. We found a weak charge state dependence of the K D for both protein-ligand systems, where for all ionization techniques the K D value decreases with increasing charge state. We demonstrate that ESSI is very gentle and insensitive to instrumental parameters, and the K D obtained is in good agreement with solution phase results from the literature. In addition, we tried to determine the K D for the lymphocyte-specific kinase LCK binding to a kinase inhibitor using nanoESI due to the very low amount of sample available. In this case, we found K D values with a strong charge state dependence, which were in no case close to literature values for solution phas

How to deal with weak interactions in noncovalent complexes analyzed by electrospray mass spectrometry: Cyclopeptidic inhibitors of the nuclear receptor coactivator 1-STAT6

Mass spectrometry, and especially electrospray ionization, is now an efficient tool to study noncovalent interactions between proteins and inhibitors. It is used here to study the interaction of some weak inhibitors with the NCoA-1/STAT6 protein with K D values in the µM range. High signal intensities corresponding to some nonspecific electrostatic interactions between NCoA-1 and the oppositely charged inhibitors were observed by nanoelectrospray mass spectrometry, due to the use of high ligand concentrations. Diverse strategies have already been developed to deal with nonspecific interactions, such as controlled dissociation in the gas phase, mathematical modeling, or the use of a reference protein to monitor the appearance of nonspecific complexes. We demonstrate here that this last methodology, validated only in the case of neutral sugar-protein interactions, i.e., where dipole-dipole interactions are crucial, is not relevant in the case of strong electrostatic interactions. Thus, we developed a novel strategy based on half-maximal inhibitory concentration (IC50) measurements in a competitive assay with readout by nanoelectrospray mass spectrometry. IC50 values determined by MS were finally converted into dissociation constants that showed very good agreement with values determined in the liquid phase using a fluorescence polarization assa

First report of fatty acid-derived alkaloids produced by species of the ladybird genus Scymnus (Coccinellidae: Coleoptera)

ABSTRACT: Species in the genus Scymnus Kugelann are common, but there is little information available on their defence compounds. Pupae are covered with setae on tips of which there are small droplets of liquid. This study explored the patterns of the distribution of the droplets and the influence of diet on their production in five species of Scymnus. GC-MS analyses were used to determine the chemical identity of droplets. All these species produced droplets, but the patterns in the distributions varied among species. Preliminary data indicates a de novo synthesis of the droplet compounds by these ladybirds. The results of the GC-MS analyses suggest that Scymnus spp. produce azamacrolides, which are fatty acid-derived alkaloids.info:eu-repo/semantics/publishedVersio

In vivo monitoring of the corneal collagen cross-linking using supersonic shear wave imaging: feasibility study on porcine corneas

International audienceThe cornea is the main refractive component of the eye. Defects in corneal shape can reduce the visual acuity. Corneal ectasia is a case of severe deformation, which is characterized by a progressive bulging. It can occur naturally or after LASIK surgery, leading to damaged vision and ultimately blindness. Corneal cross-linking (CXL) has recently been proposed as a minimally invasive treatment to stop the disease progression. It consists in photo-reticulating the collagen fibrils to stiffen the cornea. However, this treatment is currently not monitored in real-time. Here, we propose Supersonic Shear Wave Imaging (SSI) to measure the stiffening effect of CXL. SSI elastography consists in generating and tracking a shear wave in tissues using ultrafast (30000 frames/sec) ultrasonic scanners (Aixplorer, SuperSonic Imagine). The tissue elasticity is deduced from the shear wave speed. For corneal applications, we implemented SSI on high-frequency ultrasonic arrays (15MHz). We performed in vivo CXL on anesthetized pigs combined with SSI monitoring. We obtained elastic maps after CXL that exhibited significant stiffening in the treated area (56 ± 15% of the shear wave speed) compared to the untreated area. These results demonstrated the feasibility of SSI for the in vivo and real-time monitoring of CXL

One-Year Risk of Stroke after Transient Ischemic Attack or Minor Stroke

BACKGROUND Previous studies conducted between 1997 and 2003 estimated that the risk of stroke or an acute coronary syndrome was 12 to 20% during the first 3 months after a transient ischemic attack (TIA) or minor stroke. The TIAregistry.org project was designed to describe the contemporary profile, etiologic factors, and outcomes in patients with a TIA or minor ischemic stroke who receive care in health systems that now offer urgent evaluation by stroke specialists. METHODS We recruited patients who had had a TIA or minor stroke within the previous 7 days. Sites were selected if they had systems dedicated to urgent evaluation of patients with TIA. We estimated the 1-year risk of stroke and of the composite outcome of stroke, an acute coronary syndrome, or death from cardiovascular causes. We also examined the association of the ABCD2 score for the risk of stroke (range, 0 [lowest risk] to 7 [highest risk]), findings on brain imaging, and cause of TIA or minor stroke with the risk of recurrent stroke over a period of 1 year. RESULTS From 2009 through 2011, we enrolled 4789 patients at 61 sites in 21 countries. A total of 78.4% of the patients were evaluated by stroke specialists within 24 hours after symptom onset. A total of 33.4% of the patients had an acute brain infarction, 23.2% had at least one extracranial or intracranial stenosis of 50% or more, and 10.4% had atrial fibrillation. The Kaplan–Meier estimate of the 1-year event rate of the composite cardiovascular outcome was 6.2% (95% confidence interval, 5.5 to 7.0). Kaplan–Meier estimates of the stroke rate at days 2, 7, 30, 90, and 365 were 1.5%, 2.1%, 2.8%, 3.7%, and 5.1%, respectively. In multivariable analyses, multiple infarctions on brain imaging, large-artery atherosclerosis, and an ABCD2 score of 6 or 7 were each associated with more than a doubling of the risk of stroke. CONCLUSIONS We observed a lower risk of cardiovascular events after TIA than previously reported. The ABCD2 score, findings on brain imaging, and status with respect to large-artery atherosclerosis helped stratify the risk of recurrent stroke within 1 year after a TIA or minor stroke. (Funded by Sanofi and Bristol-Myers Squibb.)Supported by an unrestricted grant from Sanofi and Bristol-Myers Squibb