Evaluation of Jacalin lectin sorbents for the extraction of the human chorionic gonadotropin glycoforms prior to analysis by nano liquid chromatography-high resolution mass spectrometry.

Human chorionic gonadotropin (hCG) is a dimeric, highly glycosylated hormone with a total of 4 N- and 4 O-glycosylation sites in its two subunits, hCGα and hCGβ. Recently, we developed a novel nano liquid chromatography coupled to high resolution mass spectrometry (nanoLC-HRMS) method for the analysis and thus the detection of the intact glycoforms of hCG. Here, a sorbent functionalized with the Jacalin lectin was evaluated in solid-phase extraction (SPE) for its potential to fractionate the hCG glycoforms prior to their nanoLC-HRMS analysis at the intact level, which may facilitate the detection of low-abundance glycoforms and may lead to a more detailed characterization of the hormone glycosylation.

Salt assisted liquid-liquid extraction combined with dispersive liquid-liquid microextraction for the determination of 24 regulated polycyclic aromatic hydrocarbons in human serum.

Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants of great concern due to their carcinogenicity and mutagenicity. Their determination in human serum, particularly in at-risk populations, is necessary but difficult because they are distributed over a wide range of polarity and are present at trace level. A new method combining salting-out assisted liquid-liquid extraction (SALLE) and dispersive liquid-liquid microextraction with solidification of floating organic drop (DLLME-SFO) adapted to a reduced volume of sample (100 µl) was developed to determine 24 PAHs in human serum.

Use of the dummy approach for the synthesis of ion imprinted polymers with Ni(II) or Zn(II) as template ion for the solid-phase extraction of Cu(II).

There is a strong interest in monitoring copper in environmental waters, but its direct analysis suffers from strong matrix interferences. This is why, a sample pretreatment based on solid-phase extraction (SPE) is often used but conventional sorbents usually lack specificity. It is overcome with ion-imprinted polymers (IIPs).

Characterization of Concanavalin A-based lectin sorbents for the extraction of the human chorionic gonadotropin glycoforms prior to analysis by nano liquid chromatography-high resolution mass spectrometry.

Human chorionic gonadotropin (hCG) is constituted of the hCGα and hCGβ subunits and is a highly glycosylated protein. Affinity supports based on immobilized Concanavalin A (Con A) lectin were used in solid phase extraction (SPE) to fractionate the hCG glycoforms according to their glycosylation state. For the first time, the lectin SPE fractions were off-line analysed by a nano liquid chromatography - high-resolution mass spectrometry (nanoLC-HRMS) method keeping the glycoforms intact.

Novel oligonucleotide-based sorbent for the selective extraction of cadmium from serum samples.

The objective was to develop a sorbent functionalized with aptamers for the selective extraction of cadmium from biological samples. Two oligonucleotide sequences reported in literature as specific to cadmium were covalently grafted on activated Sepharose, with grafting yields of 45%. Once the supports packed in cartridges, a thorough study of the percolation conditions favoring Cd(II) retention was performed, demonstrating the importance of the nature of this medium.

Development of setups for real-time analysis of the effluent of a microreactor by mass spectrometry.

Monitoring a synthesis reaction in real time could allow not only the detection of the intermediates involved in the synthesis, to better understand its mechanisms, but also the impurities. Spectroscopic methods could be performed but are not so performant when analyzing complex mixtures and could require specific properties for the detection of the molecules of interest, the presence of a chromophore moiety for example. Mass spectrometry (MS) may overcome these limitations and is able to reach the accuracy and sensitivity required to efficiently detect, quantify, identify, and characterize the reagents and species produced during the synthesis.

Development of ion-imprinted polymers for the selective extraction of Cu(II) ions in environmental waters.

Several ion-imprinted polymers (IIPs) were synthesized via bulk polymerization with Cu(II) as template ion, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as crosslinking agent, and azobisisobutyronitrile as initiator in acetonitrile or methanol as porogen solvent. Non-imprinted polymers (NIPs) were similarly synthesized but without Cu(II). After grounding and sieving, the template ions were removed from IIPs particles through several cycles of elimination in 3 M HCl.

Ra and Cs determination by inductively coupled plasma mass spectrometry: state of the art and perspectives including sample pretreatment and separation steps.

Achieving precise and accurate quantification of radium (Ra) and cesium (Cs) by inductively coupled plasma mass spectrometry (ICP-MS) is of particular interest in the field of radiological monitoring and more widely in environmental and biological sciences. However, the accuracy and sensitivity of the quantification depend on the analytical strategy implemented. Eliminating interferences during the sample handling step and/or during the analysis step is critical since presence of matrix elements can lead to spectral and non-spectral interferences in ICP-MS.

Recent Advances in Lectin-Based Affinity Sorbents for Protein Glycosylation Studies.

Glycosylation is one of the most significant post-translational modifications occurring to proteins, since it affects some of their basic properties, such as their half-life or biological activity. The developments in analytical methodologies has greatly contributed to a more comprehensive understanding of the quantitative and qualitative characteristics of the glycosylation state of proteins. Despite those advances, the difficulty of a full characterization of glycosylation still remains, mainly due to the complexity of the glycoprotein and/or glycopeptide mixture especially when they are present in complex biological samples.

Simplified miniaturized analytical set-up based on molecularly imprinted polymer directly coupled to UV detection for the determination of benzoylecgonine in urine.

A simple, selective, and sensitive method involving a miniaturized solid phase extraction step based on a monolithic molecularly imprinted polymer (MIP) directly coupled on-line to UV detection was developed for the determination of benzoylecgonine (BZE) in complex biological samples. Monolithic MIPs were prepared into 100 μm internal diameter fused-silica capillaries either by thermal or photopolymerization. While leading to similar selectivities with respect to BZE, photopolymerization has made it possible to produce monoliths of different lengths that can be adapted to the targeted miniaturized application.

Identification and semi-relative quantification of intact glycoforms of human chorionic gonadotropin alpha and beta subunits by nano liquid chromatography-Orbitrap mass spectrometry.

The human chorionic gonadotropin (hCG) protein belongs to a family of glycoprotein hormones called gonadotropins. It is a heterodimer made of two non-covalently linked subunits. The α-subunit structure, hCGα, has 2 N-glycosylation sites, while the beta subunit, hCGβ, has 2 N- and 4 O-glycosylation sites.

Molecularly imprinted polymers in miniaturized extraction and separation devices.

Molecularly imprinted polymers are highly selective and cost-effective materials, which have attracted significant interest in various areas such as sample pretreatment and chromatographic and electrophoretic separations. This review aims to present the state of the art concerning the miniaturization of these materials in order to meet the societal demand for reliable, fast, cheap, and solvent/sample saving analyses. The polymerization route specificities for the production of miniaturized molecularly imprinted polymers in capillaries or chip channels, such as open tubular, packed particles, magnetic nanoparticles, and in situ imprinted monoliths, are investigated.

Immunoaffinity Extraction and Alternative Approaches for the Analysis of Toxins in Environmental, Food or Biological Matrices.

The evolution of instrumentation in terms of separation and detection allowed a real improvement of the sensitivity and analysis time. However, the analysis of ultra-traces of toxins in complex samples requires often a step of purification and even preconcentration before their chromatographic analysis. Therefore, immunoaffinity sorbents based on specific antibodies thus providing a molecular recognition mechanism appear as powerful tools for the selective extraction of a target molecule and its structural analogs to obtain more reliable and sensitive quantitative analysis in environmental, food or biological matrices.

Identification and semi-relative quantification of intact glycoforms by nano-LC-(Orbitrap)MS: application to the α-subunit of human chorionic gonadotropin and follicle-stimulating hormone.

Human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH) belong to the family of glycoprotein polypeptide hormones called gonadotropins. They are heterodimers sharing the α-subunit structure that has 2 N-glycosylation sites. A method based on nano-reversed-phase liquid chromatography coupled to high-resolution mass spectrometry with an Orbitrap analyzer was developed for the first time to characterize the glycosylation state of the α-subunit at the intact level.

Analysis of the human chorionic gonadotropin protein at the intact level by HILIC-MS and comparison with RPLC-MS.

In the present work, the human chorionic gonadotropin (hCG) hormone was characterized for the first time by hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution (HR) quadrupole/time-of-flight (qTOF) mass spectrometry (MS) at the intact level. This heterodimeric protein, consisting of two subunits (hCGα and hCGβ), possesses 8 potential glycosylation sites leading to a high number of glycoforms and has a molecular weight of about 35 kDa. The HILIC conditions optimized in a first paper but using UV detection were applied here with MS for the analysis of two hCG-based drugs, a recombinant hCG and a hCG isolated from the urine of pregnant women.

Selective extraction of cocaine from biological samples with a miniaturized monolithic molecularly imprinted polymer and on-line analysis in nano-liquid chromatography.

We report the on-line coupling of a monolithic molecularly imprinted polymer to nano-liquid chromatography for the selective analysis of cocaine and its main metabolite, benzoylecgonine, in complex biological samples. After the screening of different synthesis conditions, a monolithic molecularly imprinted polymer was in situ synthesized into a 100 μm internal diameter fused-silica capillary using cocaine as template, methacrylic acid as functional monomer, and trimethylolpropane trimethacrylate as cross-linker. Scanning electron microscopy was used to assess the homogeneous morphology of the molecularly imprinted polymer and its permeability was measured.

Separation methods hyphenated to mass spectrometry for the characterization of the protein glycosylation at the intact level.

Glycosylation is one of the most common post-translational modifications of proteins that affects their biological activity, solubility, and half-life. Therefore, its characterization is of great interest in proteomic, particularly from a diagnostic and therapeutic point of view. However, the number and type of glycosylation sites, the degree of site occupancy and the different possible structures of glycans can lead to a very large number of isoforms for a given protein, called glycoforms.

Development of Immobilized Enzyme Reactors for the characterization of the glycosylation heterogeneity of a protein.

The mapping of post-translational modifications (PTMs) of proteins can be addressed by bottom-up proteomics strategy using proteases to achieve the enzymatic digestion of the biomolecule. Glycosylation is one of the most challenging PTM to characterize due to its large structural heterogeneity. In this work, two Immobilized Enzyme Reactors (IMERs) based on trypsin and pepsin protease were used for the first time to fasten and improve the reliability of the specific mapping of the N-glycosylation heterogeneity of glycoproteins.

First profiling in hydrophilic interaction liquid chromatography of intact human chorionic gonadotropin isoforms.

The study of glycoproteins is a rapidly growing field, which is not surprising considering that approximately 70% of human proteins are glycosylated and that numerous biological functions are associated to the glycosylation. In this work, our interest focused on the heterodimeric human Chorionic Gonadotropin (hCG) glycoprotein that is the specific hormone of the human pregnancy, consisting of an α and a β subunit, so-called hCGα and hCGβ, respectively. This protein possesses a very high structural heterogeneity, essentially due to the presence of 8 glycosylation sites, but also other types of post-translational modifications.

First characterizations by capillary electrophoresis of human Chorionic Gonadotropin at the intact level.

In the present work, the first characterizations by Capillary Electrophoresis of the human Chorionic Gonadotropin (hCG) hormone at the intact level were carried out. hCG is a hetero-dimeric glycoprotein, specific to the human pregnancy, consisting of an α and a β subunit, so-called hCGα and hCGβ, respectively. hCG has 8 potential glycosylation sites leading to a high number of isoforms (including glycoforms and other post-translational modifications) that we are interesting to characterize.

An attempt to characterize the human Chorionic Gonadotropin protein by reversed phase liquid chromatography coupled with high-resolution mass spectrometry at the intact level.

For the first time, the human Chorionic Gonadotropin (hCG) hormone at the intact level was characterized by reversed phase liquid chromatography (RPLC) coupled with high resolution mass spectrometry (HRMS). This heterodimeric protein is specific to human pregnancy, consists in an α and a β subunit, so-called hCGα and hCGβ, respectively, and has 8 glycosylation sites leading to a high number of isoforms. First, the LC method was optimized to separate the largest number of isoforms and also to facilitate the MS ionization process and data treatment.

First investigations for the characterization of glucosamine-6-phosphate synthase by capillary electrophoresis.

The enzyme glucosamine-6-phosphate synthase (GlmS) is an important point of metabolic control in biosynthesis of amino sugar-containing macromolecules and is therefore a potential target in order to design antibacterial and antifungal drugs. It has two oligomerization states, which are the active dimer and the inactive hexamer. For the first time, the potential of CE to separate and quantify the two forms was studied.

Kinetic study of niobium and tantalum hexameric forms and their substituted ions by capillary electrophoresis in alkaline medium.

In this work a capillary electrophoretic (CE) method is used for the kinetic study of the intermetallic substitutions in hexameric ions of two strategic metals, tantalum and niobium in an alkaline medium. Recently proposed processes for the production and analytical separation of tantalum and niobium that are faster, more economical and environmental friendly are based on the use of highly alkaline media. It was previously established that in these media, tantalum and niobium exist as hexameric species, HTaO (Ta) and HNb0 (Nb), which can be analysed with a CE method using an alkaline electrolyte and UV detection.