Component-resolved diagnostic (CRD) of cow's milk allergy has been performed using immunoaffinity... more Component-resolved diagnostic (CRD) of cow's milk allergy has been performed using immunoaffinity capillary electrophoresis (IACE) coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). First, total IgE quantification in the blood serum of a milk allergic patient by the IACE-UV technique was developed using magnetic beads (MBs) coated with antihuman IgE antibodies (Abs) to perform the general allergy diagnosis. Then, the immunocomplex of antihuman IgE Abs with the patient IgE Abs, obtained during the total IgE analysis, was chemically cross-linked on the MBs surface. Prepared immunosupport was used for the binding of individual milk allergens to identify the proteins triggering the allergy by IACE with UV and MALDI MS detection. Then, allergy CRD was also performed directly with milk fractions. Bovine serum albumin, lactoferrin, and α-casein (S1 and S2 forms, as was revealed by MALDI MS) were found to bind with the extracted IgE Abs, indicating that the chosen patient is allergic to these proteins. The results were confirmed by performing classical enzyme-linked immunosorbent assay of total and specific IgE Abs. The present IACE-UV/MALDI MS method required only 2 μL of blood serum and allowed the performance of the total IgE quantification and CRD of the food allergy not only with the purified allergen molecules but also directly with the food extract. Such an approach opens the possibility for direct identification of allergens molecular mass and structure, discovery of unusual allergens, which could be useful for precise personalized allergy diagnostic, allergens epitope mapping, and cross-reactivity studies.
An electrostatic-spray ionization (ESTASI) method has been used for mass spectrometry (MS) analys... more An electrostatic-spray ionization (ESTASI) method has been used for mass spectrometry (MS) analysis of samples deposited in or on an insulating substrate. The ionization is induced by a capacitive coupling between an electrode and the sample. In practice, a metallic electrode is placed close to but not in direct contact with the sample. Upon application of a high voltage pulse to the electrode, an electrostatic charging of the sample occurs leading to a bipolar spray pulse. When the voltage is positive, the bipolar spray pulse consists first of cations and then of anions. This method has been applied to a wide range of geometries to emit ions from samples in a silica capillary, in a disposable pipet tip, in a polymer microchannel, or from samples deposited as droplets on a polymer plate. Fractions from capillary electrophoresis were collected on a polymer plate for ESTASI MS analysis.
A compatible buffer system for coupling of capillary electrophoresis (CE) with matrix-assisted la... more A compatible buffer system for coupling of capillary electrophoresis (CE) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was developed. The employed interface consists of a robot to drive a silver-covered separation capillary and an AnchorChip MALDI-MS target. The outlet of the capillary is grounded and connected to the pre-deposited buffer droplet on the MALDI target to make the electric connection and allow sample crystallization for MALDI-MS. The possibility of using only one buffer already containing the matrix for MALDI-MS for separation and ionization was investigated and tested on protein and peptide samples. The results show that the proposed buffer system is suitable for CE-MALDI-MS coupling, simplifies the traditional buffer mixing steps in off-line CE-MALDI-MS protocols, and is therefore highly promising for on-line analysis.
The main scientific objective of the project is to promote and facilitate proteomics studies by d... more The main scientific objective of the project is to promote and facilitate proteomics studies by developing new technologies for the analysis of proteins in complex mixtures, that are complementary to conventional 2-dimensional gel electrophoresis. To reach this goal, the design, analytical development and industrial fabrication of a complete platform is pursued comprising a new generation of automated microand nano-scale proteomics systems for isolation, purification and structural characterisation of proteins from complex biological mixtures. Key experimental approaches and methods are (i) the development of new pre-fractionation microelectrolysers in miniaturised format to fit the requirements of microfluidic systems; (ii) synthesis of new wall-coated polymers for use in fused silica capillaries, and of thermosensitive matrices suitable for proteomics microchips; (iii) design and fabrication of polymeric microchips with performance characteristics, suitable for general microfluidi...
The definition of a representative control group was challenging. The risks attributed to thymect... more The definition of a representative control group was challenging. The risks attributed to thymectomy can be regarded as superimposed on risks related to the underlying cardiac defect. To distinguish the effects of thymectomy from the underlying heart defect, we used 2 different control groups. Individuals in the surgery control group underwent cardiac surgery before the age of 5 years, but the type of surgery did not involve thymectomy. Inherently the thymectomized individuals and the surgery controls do not share underlying cardiac defects, a fact that compromises comparability. The nationwide Swedish registers enable follow-up with minimal losses. 8 Nonetheless an ascertainment bias cannot be excluded because thymectomized persons are perhaps more likely than the general population to be managed at hospital clinics, with excellent register coverage, than by private caregivers where coverage is uncertain, or in primary health care without register coverage. However, the surgery control group is probably similar to thymectomized persons in this respect. Early thymectomy leads to immunological changes reminiscent of those accompanied by aging. 9 Possible clinical consequences of thymectomy do not necessarily become overt until later in life and therefore it is important to take into consideration the relatively young age and short follow-up time of most of the individuals included in this study. Their ages at the end of the study range from 1 to 37 years (mean, 14 years) and the mean follow-up time was 13 years. This study detects risk differences for certain autoimmune diseases, cancer, infections, and asthma following early thymectomy. Because of the observational nature of this study, it was not possible to ascertain a causal mechanism. Even so, thymectomy must be regarded as a potential explanation for the findings, and we find it advisable during early cardiac surgery to avoid total thymectomy if possible.
TiO2-facilitated MALDI–TOF-MS was proposed to improve intact bacteria fingerprinting, allowing ra... more TiO2-facilitated MALDI–TOF-MS was proposed to improve intact bacteria fingerprinting, allowing rapid and convenient antimicrobial resistance-associated protein detection during bacteria identification.
A limited amount and extreme concentration variability of proteomic-related samples require effic... more A limited amount and extreme concentration variability of proteomic-related samples require efficient analyte preconcentration and purification prior to the mass spectrometry (MS)-based analysis. Preferably, these steps should be coupled online with chosen fractionation and detection techniques for the minimization of the sample loss. To realize such sample pretreatment, herein, an on-chip solid-phase extraction-gradient elution-tandem mass spectrometry (SPE-GEMS/MS) is introduced. This technique combines in a microfluidic format online sample preconcentration/purification on SPE sorbent with further fractionation and MS/MS analysis. C8-functionalized mesoporous magnetic microspheres are chosen as a sorbent, spatially confined with an applied magnetic field. They ensure a selective enrichment and analysis of large hydrophobic peptides (2.5-7 kDa), matching the desired mass bin of the extended bottom-up proteomic (eBUP, 3-7 kDa) approach. Within less than 35 min and without additiona...
For effective monitoring and prevention of the food allergy, one of the emerging health problems ... more For effective monitoring and prevention of the food allergy, one of the emerging health problems nowadays, existing diagnostic procedures and allergen detection techniques are constantly improved. Meanwhile, new methods are also developed, and more and more putative allergens are discovered. This review describes traditional methods and summarizes recent advances in the fast evolving field of the in vitro food allergy diagnosis, allergen detection in food products and discovery of the new allergenic molecules. A special attention is paid to the new diagnostic methods under laboratory development like various immuno- and aptamer-based assays, including immunoaffinity capillary electrophoresis. The latter technique shows the importance of MS application not only for the allergen detection but also for the allergy diagnosis.
An advanced approach is developed in this work for simultaneous on-line separation and digestion ... more An advanced approach is developed in this work for simultaneous on-line separation and digestion of proteins by combining the Off-Gel isoelectric focusing (IEF) and enzymatic nanoreactor enhanced proteolysis. The nanoreactor was prepared by preloading trypsin in amino-functionalized macroporous silica, and then directly added into Off-Gel wells. With the nanoreactor loaded Off-Gel device, effective digestion of proteins happened during IEF electrophoresis to generate directly fractionated tryptic peptides, which not only accelerated the experimental flow but also avoided sample loss, leading to a more comprehensive protein identification from complex biological samples. A successful identification of 3592 proteins was achieved from Hela cell line by using the approach followed with LC-MS/MS analysis, while only 1877 proteins were identified from the same sample when using standard in-solution proteolysis followed with Off-Gel electrophoresis and then LC-MS/MS analysis. Therefore, we have demonstrated that the approach can greatly simplify high-throughput proteomics and significantly improve protein identification.
Component-resolved diagnostic (CRD) of cow's milk allergy has been performed using immunoaffi... more Component-resolved diagnostic (CRD) of cow's milk allergy has been performed using immunoaffinity capillary electrophoresis (IACE) coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). First, total IgE quantification in the blood serum of a milk allergic patient by the IACE-UV technique was developed using magnetic beads (MBs) coated with antihuman IgE antibodies (Abs) to perform the general allergy diagnosis. Then, the immunocomplex of antihuman IgE Abs with the patient IgE Abs, obtained during the total IgE analysis, was chemically cross-linked on the MBs surface. Prepared immunosupport was used for the binding of individual milk allergens to identify the proteins triggering the allergy by IACE with UV and MALDI MS detection. Then, allergy CRD was also performed directly with milk fractions. Bovine serum albumin, lactoferrin, and α-casein (S1 and S2 forms, as was revealed by MALDI MS) were found to bind with the extracted IgE Abs, indicating th...
β‐Ga2O3 is a wide‐band‐gap semiconductor having strong oxidation ability under light irradiation.... more β‐Ga2O3 is a wide‐band‐gap semiconductor having strong oxidation ability under light irradiation. Herein, the steel target plates modified with β‐Ga2O3 nanoparticles have been developed to carry out in‐source photo‐catalytic oxidative reactions for online peptide tagging during laser desorption/ionization mass spectrometry (LDI‐MS) analysis. Under UV laser irradiation, β‐Ga2O3 can catalyze the photo‐oxidation of 2‐methoxyhydroquinone added to a sample mixture to 2‐methoxy benzoquinone that can further react with the thiol groups of cysteine residues by Michael addition reaction. The tagging process leads to appearance of pairs of peaks with an m/z shift of 138.1Th. This online labelling strategy is demonstrated to be sensitive and efficient with a detection‐limit at femtomole level. Using the strategy, the information on cysteine content in peptides can be obtained together with peptide mass, therefore constraining the database searching for an advanced identification of cysteine‐co...
A versatile microreactor protocol based on microfluidic droplets has been developed for on-line p... more A versatile microreactor protocol based on microfluidic droplets has been developed for on-line protein digestion. Proteins separated by liquid chromatography are fractionated in water-in-oil droplets and digested in sequence. The microfluidic reactor acts also as an electrospray ionization emitter for mass spectrometry analysis of the peptides produced in the individual droplets. Each droplet is an enzymatic microreaction unit with efficient proteolysis due to rapid mixing, enhanced mass transfer and automated handling. This droplet approach eliminates sample loss, cross-contamination, nonspecific absorption and memory effect. A protein mixture was successfully identified using the droplet-based micro-reactor as interface between reverse phase liquid chromatography and mass spectrometry. During the last decade, two approaches have been developed for proteomics research: top-down and bottom-up. Among the various strategies, shotgun proteomics has been widely used because of its high throughput and automation. The complex samples are enzymatically digested and then separated by high performance liquid chromatography (HPLC), before being analysed by mass spectrometry (MS) for sequencing via database searching. However, it is difficult to achieve the simultaneous digestion of large amount of proteins, the separation of thousands peptides and data processing for accurate protein identification. Peptides from high-abundance proteins may undermine the detection of those from low-abundance ones. Therefore, if the protein mixture is completely pre-separated, where a simple protein component is found in each fraction, and followed with in situ digestion, more detailed information about protein sequence and post-translational modification would be obtained. Recently, many strategies have been developed for proteomics through protein separation, such as Offgel electrophoresis, 5 proteolysis, peptide separation and MS detection. The development of integrated platforms to achieve automatic operation of the whole systems have been proposed for high throughput characterization of proteins. 9,10 For example, Zhang et al. have established an integrated platform with the combination of protein separation by liquid chromatography (LC), on-line protein digestion by immobilized trypsin column, peptides separation and peptide identification by electrospray ionization mass spectrometry (ESI-MS/MS). The immobilized trypsin has been demonstrated to be a highly efficient protocol for on-line digesting proteins. 11-14 However, the micro-reactors have the drawback of non-specific absorption of proteins and peptides resulting in a memory effect, which hampers the large-scale proteome study. It is also difficult to collect fractions for storage and further protein characterization. Compartmentalization of effluents into droplets with microfluidic chip has emerged as a novel way to collect fractions from separation methods such as HPLC and CE. Droplet-based microfluidic technique offers an attractive alternative to conventional microfluidic chips based on continuous flow systems by performing chemical and biological reactions in picoliter to nanoliter volumes without dilution and cross-contamination. In essence, each droplet is analogous to a conventional reaction unit but with the advantages of the following: reduced reagent consumption, rapid mixing, enhanced diffusion, efficient heat and mass transfer, automated handling and continuous processing. Meanwhile, mass spectrometry is the most attractive analytical technique for the analysis of segmented flows, because it has the high sensitivity and speed for the analysis of low volume samples at high throughput. Recent advances in mass spectrometry 22 and microfluidics 23 have enabled integration of these tools to address the requirement of high-throughput protein analysis. Based on these ideas, we present a droplet-based microfluidic reactor for online tryptic digestion of proteins from HPLC fractions for the first time. The generated peptides were either collected for matrix-assisted laser desorption/ionization (MALDI) MS analysis or directly electro-sprayed from the outlet of the microchip for MS identification. In this way, the proposed droplet micro-reactor can be used as an interface between protein separation by liquid chromatography and peptide analysis by
Journal of the American Society for Mass Spectrometry, 2013
A dual-channel electrospray microchip has been developed for electrospray ionization mass spectro... more A dual-channel electrospray microchip has been developed for electrospray ionization mass spectrometry (ESI-MS) where aqueous samples are mixed at the Taylor cone with an organic buffer. Due to the fast and effective mixing in the Taylor cone, the aqueous sample can be well ionized with a high ion intensity. The influence of geometric parameters such as the distance between the two microchannels at their junction at the tip of the emitter has been investigated together with chemical parameters such as the organic buffer composition.
An on-plate specific enrichment method is presented for the direct analysis of peptides phosphory... more An on-plate specific enrichment method is presented for the direct analysis of peptides phosphorylation. An array of sintered TiO 2 nanoparticle spots was prepared on a stainless steel plate to provide porous substrate with a very large specific surface and durable functions. These spots were used to selectively capture phosphorylated peptides from peptide mixtures, and the immobilized phosphopeptides could then be analyzed directly by MALDI MS after washing away the nonphosphorylated peptides. β-Casein and protein mixtures were employed as model samples to investigate the selection efficiency. In this strategy, the steps of phosphopeptide capture, purification, and subsequent mass spectrometry analysis are all successfully accomplished on a single target plate, which greatly reduces sample loss and simplifies analytical procedures. The low detection limit, small sample size, and rapid selective entrapment show that this on-plate strategy is promising for online enrichment of phosphopeptides, which is essential for the analysis of minute amount of samples in high-throughput proteome research.
Two major milk whey proteins, -lactoglobulin and ␣-lactalbumin, are among the main cow milk alle... more Two major milk whey proteins, -lactoglobulin and ␣-lactalbumin, are among the main cow milk allergens and can cause allergy even at a very low concentrations. Therefore, these proteins are interesting targets in food analysis, not only for food quality control but also for highlighting the presence of allergens. Herein, a sensitive analysis for -lactoglobulin and ␣-lactalbumin was developed using immunoaffinity capillary electrophoresis hyphenated with MALDI-MS. Magnetic beads functionalized with appropriate antibodies were used for -lactoglobulin and ␣-lactalbumin immunocapture inside the capillary. After elution from the beads, analyte focusing and separation were performed by transient isotachophoresis followed by MALDI-MS analysis performed through an automated iontophoretic fraction collection interface. A LOD in the low nanomolar range was attained for both whey proteins. The method developed was further applied to the analysis of different milk samples including fortified soy milk.
There has been a recent trend towards the miniaturization of analytical tools, but what are the a... more There has been a recent trend towards the miniaturization of analytical tools, but what are the advantages of microfluidic devices and when is their use appropriate? Recent advances in the field of micro-analytical systems can be classified according to instrument performance (which refers here to the desired property of the analytical tool of interest) and two important features specifically related to miniaturisation, namely reduction of the sample volume and the time-to-result. Here we discuss the contribution of these different parameters and aim to highlight the factors of choice in the development and use of microfluidic devices dedicated to protein analysis.
The present work shows that the electrochemical properties of electrospray ionization (ESI) can b... more The present work shows that the electrochemical properties of electrospray ionization (ESI) can be used to add functions to the process. As example, we show how the choice of the electrode material can be used to study interactions between metal ions and biomolecules in mass spectrometry (MS). In positive ionization MS, an electrospray device acts as anode, which implies oxidation reactions. Sacrificial electrodes (made of copper or zinc) are used to supply the electrospray current and to produce cations that are able to react on-line with compounds of interest. Thus, the interactions between copper ions and ligands or peptides were investigated by using a copper electrode. Another example is the in situ electrogeneration of a dinuclear zinc(II) complex for the mass tagging of phosphopeptides when working with a zinc electrode. In order to perform these reactions on the same microchip, a dual-channel microsprayer was used, where one channel was dedicated to the tag electrogeneration...
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Papers by Hubert Girault