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Immunoglobulin G (IgG) purification is a critical process for evaluating its role in autoimmune diseases, which are defined by the occurrence of autoantibodies. Affinity chromatography with protein G is widely considered to be the optimal technique for laboratory-scale purification. However, this technique has some limitations, including the exposure of IgG to low pH, which can compromise the quality of the purified IgG. Here, we show that alternative methods for IgG purification are possible while maintaining the quality of IgG. Different techniques for IgG purification from serum were evaluated and compared with protein G-based approaches: Melon Gel, caprylic acid-ammonium sulfate (CAAS) precipitation, anion-exchange chromatography with diethylamino ethyl (DEAE) following ammonium sulfate (AS) precipitation, and AS precipitation alone. The results demonstrated that the purification yield of these techniques surpassed that of protein G. However, differences in the purity of IgG were observed using GeLC-MS/MS. The avidity of purified IgG against selected targets (SARS-CoV-2 and topoisomerase-I) was similar between purified IgG obtained using all techniques and unpurified sera. Our work provides valuable insights for future studies of IgG function by recommending alternative purification methods that offer advantages in terms of yield, time efficiency, cost-effectiveness, and milder pH conditions than protein G.

Collagen from paleontological bones is an important organic material for isotopic measurement, radiocarbon analysis, and paleoproteomic analysis to provide information on diet, dating, taxonomy, and phylogeny. Current paleoproteomic methods are destructive and require from a few milligrams to several tens of milligrams of bone for analysis. In many cultures, bones are raw materials for artifacts that are conserved in museums, which hampers damage to these precious objects during sampling. Here, we describe a low-invasive sampling method that identifies collagen, taxonomy, and post-translational modifications from Holocene and Upper Pleistocene bones dated to 130,000 and 150 BC using dermatological skin tape discs for sampling. The sampled bone micropowders were digested following our highly optimized enhanced filter-aided sample preparation protocol and then analyzed by MALDI FTICR MS and LC-MS/MS for identifying the genus taxa of the bones. We show that this low-invasive sampling does not deteriorate the bones and achieves results similar to those obtained by more destructive sampling. Moreover, this sampling method can be carried out at archeological sites or in museums.

Peptide mass fingerprinting (PMF) using MALDI-TOF mass spectrometry allows the identification of bone species based on their type I collagen sequence. In the archaeological or paleontological field, PMF is known as zooarchaeology mass spectrometry (ZooMS) and is widely implemented to find markers for most species, including the extinct ones. In addition to the identification of bone species, ZooMS enables dating estimation by measuring the deamidation value of specific peptides. Herein, we report several enhancements to the classical ZooMS technique, which reduces to 10-fold the required bone sample amount (down to the milligram scale) and achieves robust deamidation value calculation in a high-throughput manner. These improvements rely on a 96-well plate samples preparation, a careful optimization of collagen extraction and digestion to avoid spurious post-translational modification production, and PMF at high resolution using matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance (MALDI-FTICR) analysis. This method was applied to the identification of a hundred bones of herbivores from the Middle Paleolithic site of Caours (Somme, France) well dated from the Eemian Last Interglacial climatic optimum. The method gave reliable species identification to bones already identified by their osteomorphology, as well as to more challenging samples consisting of small or burned bone fragments. Deamidation values of bones originating from the same geological layers have a low standard deviation. The method can be applied to archaeological bone remains and offers a robust capacity to identify traditionally unidentifiable bone fragments, thus increasing the number of identified specimens and providing invaluable information in specific contexts.

The distribution of photo-crosslinkable moieties onto a protein backbone can affect a biomaterial's crosslinking behavior, and therefore also its mechanical and biological properties. A profound insight in this respect is essential for biomaterials exploited in tissue engineering and regenerative medicine. In the present work, photo-crosslinkable moieties have been introduced on the primary amine groups of: (i) a recombinant collagen peptide (RCPhC1) with a known amino acid (AA) sequence, and (ii) bovine skin collagen (COL BS) with an unknown AA sequence. The degree of substitution (DS) was quantified with two conventional techniques: an ortho-phthalic dialdehyde (OPA) assay and 1H NMR spectroscopy. However, neither of both provides information on the exact type and location of the modified AAs. Therefore, for the first time, proteomic analysis was evaluated herein as a tool to identify functionalized AAs as well as the exact position of photo-crosslinkable moieties along the AA sequence, thereby enabling an in-depth, unprecedented characterization of functionalized photo-crosslinkable biopolymers. Moreover, our strategy enabled to visualize the spatial distribution of the modifications within the overall structure of the protein. Proteomics has proven to provide unprecedented insight in the distribution of photo-crosslinkable moieties along the protein backbone, undoubtedly contributing to superior functional biomaterial design to serve regenerative medicine.

Polybutadiene (PB) and polyisoprene (PI), the two most common polydienes (PD), are involved in a large number of materials and used in a wide variety of applications. The characterization of these polymers by mass spectrometry (MS) continues to be very challenging due to their high insolubility and the difficulty to ionize them. In this work, a cross-metathesis reaction was used to generate end-functionalized acetoxy ionizable oligomers for the structural deciphering of different commercial PB and PI samples. A cross-metathesis reaction was carried out between polymers and the Z-1,4-diacetoxy-2-butene as a chain transfer agent in dichloromethane using a Hoveyda–Grubbs second-generation catalyst. Well-defined acetoxy telechelic structures were obtained and analyzed by Fourier transform ion cyclotron resonance (FTICR) high-resolution MS. However, after depolymerization, low molar mass polyolefins contained some units with different configurations, suggesting an olefin isomerization reaction due to the decomposition of the catalyst. The addition of an electron-deficient reagent such as 2,6-dichloro-1,4-benzoquinone suppressed this isomerization in the case of both Z- and E-PB and PI. Ion mobility spectrometry-mass spectrometry (IMS-MS) and energy-resolved tandem mass spectrometry (ERMS) analyses confirmed a successful isomerization suppression. For comparing the results obtained by depolymerization with classical methods for polymer analysis, pyrolysis-comprehensive two-dimensional gas chromatography/mass spectrometry (Py-GC × GC–MS), atmospheric solid analysis probe (ASAP), and direct inlet probe-atmospheric pressure chemical ionization (DIP-APCI) analyses were performed on the same polymers. This strategy can be applied on a variety of synthetic and natural not yet characterized polymers.

The in-gel detection of proteins for various proteomic experiments is commonly done with the fluorescent RuIItris(bathophenanthroline disulfonate) complex (Ru(BPS)3), which is more cost-effective compared to commercial Ru-based formulations but requires tedious procedures for its preparation and strongly acidic staining conditions. Herein, we report the synthesis and characterization of heteroleptic RuIIcomplexes Ru(BPS)2(BP) and Ru(BPS)(BP)2containing bathophenanthroline (BP) and bathophenanthroline disulfonate disodium salt (BPS) in comparison with Ru(BPS)3. It was shown by fluorescent and UV–vis measurements that novel RuIIcomplexes were excitable in both UV and visible light, close to emission bands of classical lasers, which is important for successful in-gel protein detection. Novel fluorescent dyes demonstrated improved protein detection in comparison with commercially available SYPRO Ruby staining solution. In addition, unlike commonly used staining protocols, staining with Ru(BPS)(BP)2can be performed at nearly neutral pH, thereby reducing artificial post-translational modifications (PTMs).

A simple continuous flow process for aerobic oxidation of benzylic organotrifluoroborates salt by photoredox catalysis under UV irradiation is reported. Using this approach benzylic organotrifluoroborates salts could be easily oxidized to the corresponding benzaldehydes. Good to excellent yields were obtained by applying short residence times in combination with molecular oxygen as oxidant under Taylor flow configuration.

Owing to the dissemination of antibiotic resistance among Staphylococcus aureusbacteria, it is critical to find out new, safe, and effective alternatives. Plants are a major source of many bioactive compounds that have a wide range of pharmacological activities. Quercetin is an efficient plant flavonoid that has many pharmacological effects such as the anti-microbial potential. Unfortunately, its applicability is limited due to its poor solubility, limited bioavailability and instability. Nano-drug delivery could improve efficiently drug solubility and bioavailability. The inclusion of natural components in the manufacture of nano-carriers improves their safety and biocompatibility. The aim of the present study is the fabrication of green nanoparticles of the natural bioactive, quercetin, using natural Prunus armeniacagum exudate (PAGE) as a nano-carrier. The gum composition was characterized by a glycoproteomic study which revealed the presence of polysaccharide elements from the arabinogalactan family. It also indicated chemical modifications of the gum by oxidation and the existence of many proteins involved in the plant's defense against environmental stress. The development of quercetin-loaded PAGE-NPs was based on the electrostatic interactions of calcium ions with PAGE. The formulation process was based on 23factorial design. After examining how the independent factors significantly affected drug encapsulation efficiency (DEE%) and % quercetin released after 24 h (Q24h), F6 was chosen as the optimized PAGE-NPs. F6 showed significant improvement in the intestinal permeation and stability than the unprocessed quercetin. Regarding the anti-bacterial effect, there was an improvement in the histological features in the PAGE NPs-treated group. Also, there was a significant decline in the bacterial load and enhancement of the survival rate in this group. In addition, the inflammatory mediators (IL-1β and IL-6) substantially diminished in PAGE NPs-treated group. Consequently, PAGE-NPs are efficient green nano-drug delivery systems for enhancing the pharmacological effect of quercetin.

Skeletal muscle, the most abundant body tissue, plays vital roles in locomotion and metabolism. Myostatin is a negative regulator of skeletal muscle mass. In addition to increasing muscle mass, Myostatin inhibition impacts muscle contractility and energy metabolism. To decipher the mechanisms of action of the Myostatin inhibitors, we used proteomic and transcriptomic approaches to investigate the changes induced in skeletal muscles of transgenic mice overexpressing Follistatin, a physiological Myostatin inhibitor. Our proteomic workflow included a fractionation step to identify weakly expressed proteins and a comparison of fast versus slow muscles. Functional annotation of altered proteins supports the phenotypic changes induced by Myostatin inhibition, including modifications in energy metabolism, fiber type, insulin and calcium signaling, as well as membrane repair and regeneration. Less than 10% of the differentially expressed proteins were found to be also regulated at the mRNA level but the Biological Process annotation, and the KEGG pathways analysis of transcriptomic results shows a great concordance with the proteomic data. Thus this study describes the most extensive omics analysis of muscle overexpressing Follistatin, providing molecular-level insights to explain the observed muscle phenotypic changes.

The esterification reaction is one of the most important reaction in organic chemistry, and the development of scalable continuous flow conditions is of high interest. Here we describe a scaling-up study of benzoic acid alkylation by MeI in the presence of superbases from a capillary reactor (282 mg·h–1at 75 °C) to a low flow reactor (LFR, 6.82 g·h–1at 80 °C). First, the optimization of the base demonstrated that 1,8-diazabicyclo[5.4.0]-undec-7-ene (DBU) was a good compromise in terms of basicity and price. A comparison between batch and LFR reaction showed similar kinetic rates. We also observed a maximum reactivity for a flow rate of 3 mL·min–1which corresponds to an optimum mixing of the entering fluids. A colorimetric study permits to show a visual change in the flow regime (dispersed to stratified) by varying the flow rate with a maximum conversion obtained at 3 mL·min–1corresponding to a 105 s residence time, demonstrating that the studied reaction is sensitive to mixing conditions and that LFR presents some mixing limitations for low flow rates. After thermal optimization (80 °C), the reaction has been improved to a productivity of 6.82 g/h at the lab scale using DBU as the base and Eyring/Arrhenius plots permitted to extract activation energies.

During storage, a series of changes occur for dairy powders, such as protein lactosylation and the formation of Maillard reaction products (MRPs), leading to powder browning and an increase of insoluble matter. The kinetics of protein lactosylation and MRP formation are influenced by the lactose content of the dairy powder. However, the influence of lactose in the formation of insoluble matter and its role in the underlying mechanisms is still a subject of speculation. In this study, we aim to investigate the role of lactose in the formation of insoluble matter in a more comprehensive way than the existing literature. For that, two casein powders with radically different lactose contents, standard micellar casein (MC) powder (MC1) and a lactose-free (less than 10 ppm) MC powder (MC2), were prepared and stored under controlled conditions for different periods of time. Powder browning index measurements and solubility tests on reconstituted powders were performed to study the evolution of the functional properties of MC powders during aging. Proteomic approaches [one-dimensional electrophoresis and liquid chromatography–mass spectrometry (LC–MS)] and innovative label-free quantification methods were used to track and quantify the chemical modifications occurring during the storage of the powders. Reducing the amount of lactose limited the browning of MC powders but had no effect on the loss of solubility of proteins after storage, suggesting that the action of lactose, leading to the production of MRC, does not promotes the formation of insoluble matter. Electrophoresis analysis did not reveal any links between the formation of covalent bonds between caseins and loss in solubility, regardless of the lactose content. However, LC–MS analyses have shown that different levels of chemical modifications occur during the MC powder storage, depending upon the presence of lactose. An increase of protein lactosylation and acetylation was observed for the powder with a higher lactose content, while an increase of protein deamidation and dephosphorylation was observed for that containing lower lactose. The decrease of pH in the presence of lactose as a result of Maillard reaction (MR) may explain the difference in the chemical modifications of the two powders. In view of the present results, it is clear that lactose is not a key factor promoting insolubility and for the formation of cross-links between caseins during storage. This suggests that lactosylation is not the core reaction giving rise to loss in solubility.

Ce travail porte sur la conception de microsystèmes microfluidiques dédiés à l’analyse de protéines par spectrométrie de masse ESI (ESI-MS). Dans un premier temps, nous présentons la filière technologique multi-matériaux développée. Elle répond à plusieurs contraintes, dont les principales sont liées à l’architecture et à la compatibilité avec l’application visée. Dans un second temps, nous présentons un prototype qui comprend un microcanal fonctionnalisable couplé à une interface d’electronébulisation nécessaire à l’alimentation du spectromètre de masse. Enfin deux types de caractérisation du prototype sont illustrés :

Ce travail vise à illustrer les apports potentiels de la microfluidique pour des applications de chimie organique. L’étude a été menée sur une réaction bien connue au laboratoire, la réaction de méthylation d’un polyphénol, la quercétine. Cette réaction donne lieu en conditions classiques à un mélange complexe de plusieurs produits de méthylation, sur les différentes positions phénols de la molécule et avec divers degrés de méthylation. Nous avons ainsi comparé les résultats obtenus, soit en utilisant de la verrerie classique soit dans un micro-réacteur constitué d’un mélangeur commercial (Nanomixer™, Upchurch Scientific) placé en amont d’un capillaire (d.i. 75 µm). Il ressort de cette étude que la réaction est mieux contrôlée à l’échelle microfluidique ; elle devient même sélective. Ceci s’explique en partie par la qualité du mélange obtenu à cette échelle.

This work aims at developing novel nanoESI emitter tips which are compatible with automated analysis and the use of a robotics interface and which allow for enhancing analysis conditions using electrospray ionization-mass spectrometry techniques (ESI-MS). Our novel sources borrow much from calligraphy quills in their geometry and functioning principle. The device is planar and relies on a capillary slot that guides fluids to their ionization point. Different series of nib prototypes allowed us to optimize their ionization performances, the first ones being fabricated in the negative photoresist SU-8 and the last ones in silicon-based materials. The successive series of nib emitter tips were tested on an ion trap mass spectrometer using various types of samples, standard peptides, small organic species or digested or entire proteins.

Ce travail porte sur la conception de microsystèmes microfluidiques dédiés à l’analyse de protéines par spectrométrie de masse ESI (ESI-MS). Dans un premier temps, nous présentons la filière technologique multi-matériaux développée. Elle répond à plusieurs contraintes, dont les principales sont liées à l’architecture et à la compatibilité avec l’application visée. Dans un second temps, nous présentons un prototype qui comprend un microcanal fonctionnalisable couplé à une interface d’electronébulisation nécessaire à l’alimentation du spectromètre de masse. Enfin deux types de caractérisation du prototype sont illustrés : -des tests d’étanchéité du microsystème ont été menés avec succès jusqu’à une pression de 50 bar- un couplage entre une alimentation continue issue d’un microcanal et une source d’électronébulisation a été validé par spectrométrie de masse.

Ce travail vise à illustrer les apports potentiels de la microfluidique pour des applications de chimie organique. L’étude a été menée sur une réaction bien connue au laboratoire, la réaction de méthylation d’un polyphénol, la quercétine. Cette réaction donne lieu en conditions classiques à un mélange complexe de plusieurs produits de méthylation, sur les différentes positions phénols de la molécule et avec divers degrés de méthylation. Nous avons ainsi comparé les résultats obtenus, soit en utilisant de la verrerie classique soit dans un micro-réacteur constitué d’un mélangeur commercial (Nanomixer™, Upchurch Scientific) placé en amont d’un capillaire (d.i. 75 µm). Il ressort de cette étude que la réaction est mieux contrôlée à l’échelle microfluidique ; elle devient même sélective. Ceci s’explique en partie par la qualité du mélange obtenu à cette échelle.

The idea of a novel two-dimensional (2D) nanoelectrospray ionization emitter tip with the shape of a nib is explored here. This novel planar design is studied as an alternative to the needle-like standard emitter tips that suffer from a lack of reproducibility and robustness and from an inherent incompatibility with high-throughput analysis. The composition of the micro-nib sources is analogous to the working of a simple fountain pen, with a liquid reservoir linked to a micro-nib tip from which the sample is electrosprayed via a capillary slot. The micro-nib prototypes described here were fabricated using microtechnology techniques and using the epoxy-based negative photoresist SU-8. The resulting free-standing micro-nib structure was supported by a silicon wafer. We present here two series of such micro-nib sources, the latter series exhibiting improved characteristics such as a 8 µm source width of the nib tip. They were tested in mass spectrometry experiments on an ion trap mass spectrometer (LCQ Deca XP+, Thermo Finnigan) using standard peptide samples having concentrations down to 1 µM and with a high voltage (HV) supply around 1 kV for the second series of micro-nib sources. In addition to the stability of the spray, the obtained mass spectra showed the reliability of these sources for peptide analysis; the signal of the spectra was as intense and the signal-to-noise ratio (S/N) as high as that obtained with the use of standard emitter tips. Copyright © 2003 John Wiley & Sons, Ltd.

We report here on the fabrication of a novel silicon microfluidic system dedicated to protein sample preparation prior to their on-line analysis by electrospray and mass spectrometry (ESI-MS). The microsystem includes several main module, such as a digestion cell, an elution column, a pumping system as well as an integrated nano-ESI source, whose realization reflects our technological choice. After a brief presentation on microsystems, we describe our work in microfluidics. the conception and the description of the different modules, the accounting for the retained technological alternatives and the first realizations.

(−)-Epigallocatechin (EGC), one of green tea polyphenols, has been shown to inhibit growth of cancer cells. However its mechanism of action is poorly known. We show here that EGC strongly inhibited the growth of breast cancer cell lines (MCF-7 and MDA-MB-231) but not that of normal breast epithelial cells. The inhibition of breast cancer cell growth was due to an induction of apoptosis, without any change in cell cycle progression. MCF-7 cells are known to express a wild-type p53 whereas MDA-MB-231 cells express a mutated p53. The fact that EGC induced apoptosis in both these cell lines suggests that the EGC-triggered apoptosis is independent of p53 status. Moreover, neutralizing antibodies against the death receptor Fas and inhibitors of caspases, such as caspase-8 and -10, efficiently inhibited the EGC-triggered apoptosis. In addition, immunoblotting revealed that EGC treatment was correlated with a decrease in Bcl-2 and an increase in Bax level. These results suggest that EGC-triggered apoptosis in breast cancer cells requires Fas signaling.

Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was used for the structural characterization and differentiation of four isomeric O-monomethylated catechins (on phenolic positions) by the analysis of the fragmentation behaviour of catechin. The catechin fragmentation routes were rationalized and it is shown that several diagnostic ions such as ^1,3A⁺, ^1,2B⁺, and ^1,4B⁺ allow the unambiguous identification of the methylated ring. The precise position of the methyl group on each ring is determined by the difference in the relative intensities of the diagnostic ions. Isomeric O-methylepicatechins were also differentiated using this methodology. Copyright © 2000 John Wiley & Sons, Ltd.

Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) was used for the structural characterization and differentiation of four isomeric O‐monomethylated catechins (on phenolic positions) by the analysis of the fragmentation behaviour of catechin. The catechin fragmentation routes were rationalized and it is shown that several diagnostic ions such as 1,3A+, 1,2B+, and 1,4B+allow the unambiguous identification of the methylated ring. The precise position of the methyl group on each ring is determined by the difference in the relative intensities of the diagnostic ions. Isomeric O‐methylepicatechins were also differentiated using this methodology. Copyright © 2000 John Wiley & Sons, Ltd.

This paper describes a new scanning mode for quadrupole mass spectrometers. In accordance with the theoretical equations of ion motion in a quadrupolar field, an alternative method of scanning is possible varying only the RF applied to the rods of the quadrupole, keeping constant the amplitude of the AC and DC potentials. First results are reported for a RF scanning range of 0.4–1.1 MHz and an AC amplitude of approximately 250 V. © 1998 John Wiley & Sons, Ltd.

Protonated acetaldehyde is methylated on the oxygen during interaction with methanol in the gas phase. The ionic product of the ion/molecule reaction between methanol and protonated acetaldehyde is identical with C-protonated methylvinyl ether (high-pressure ionization), and with the (M − C 2H 5) +fragment ion of sec-butyl methyl ether (following electron ionization), and also with the (M − OCH 3) +fragment ion of acetaldehyde dimethylacetal (following electron ionization). The structures of these ions and the mechanism of their formation were established by isotope-labeling experiments and collision-induced dissociation mass spectra of model compounds obtained with three different types of tandem mass spectrometers (BEQQ, triple-quadrupole, and a penta-quadrupole instrument). Gas phase synthesis of the product ion from [ 2H 3]-methanol or [ 2H 4]-acetaldehyde provided insight into its mode of formation and collision-induced dissociation.

Vinyl chlorides and bromides are obtained in good yield from the corresponding ketones and acetyl halides in the presence of trifluoroacetic or trifluoromethanesulfonic acid. The Z isomer is selectively formed.

2,3,7,8,12,13,17,18‐Octafluoro‐5,10,15,20‐tetraphenylporphyrin has been prepared from 3,4‐difluoropyrrole and benzaldehyde under Lindsey conditions. An X‐ray crystal structure study of its ZnIIcomplex has shown that the macrocycle core is nonplanar, a result in apparent contradiction with a blue‐shifted UV/Vis spectrum. The results reported here demonstrate that a wide range of β‐octafluoro‐meso‐arylated porphyrins, a new class of highly electron‐deficient ligands, are potentially accessible from 3,4‐difluoropyrrole, thus opening the door to, inter alia, efficient and robust oxidation catalysts.

Summary Ab initio molecular orbital calculations give small stabilization energies for the various Na(CH4)+ adducts (less than 4 kcal mol-1), but predict a stronger binding for the copper compounds (about 13 kcal mol-1). The different behaviour of Na+ and Cu+, already present at the SCF level, is reinforced by electron correlation. This can be attributed to an important contribution of the dispersion energy to the binding energy of the copper ion: about 40% of the total, including basis set superposition corrections.

Abstract: Lignins are composed of phenylpropane units interconnected by labile and resistant bonds. A two-step degradation, thioacidolysis, provides detailed information on these network polymers. The first step involves lignin depolymerization with ethanethiol and BF3 etherate. The determination of the recovered thioethylated monomers provides an estimation of lignin units only involved in labile ether bonds. The ligninderived dimers, representative of resistant interunit bonds, are determined after a further desulfurization step. Results obtained for native and industrial hardwood lignins underline their structural differences. Native hardwood lignins are typified by a high proportion of linear fragments linked bifunctionally by β-O-4 bonds.

Ion-molecule reactivities of different one-carbon ligand complexes Fe(CX) +with oxygen and allyl chloride appeared strongly dependent on the nature and binding energy of the ligand. With oxygen ligand oxidation occurred for X = O, S. With allyl chloride the only case of the Fe(CS) +complex.