Open channel-based microchip electrophoresis interfaced with mass spectrometry via electrostatic spray ionization

The coupling between open channel-based microchip electrophoresis and mass spectrometry via electrostatic spray ionization is proposed for in situ detection of fractionated analytes. Electrophoretic separation is performed in an open channel fabricated in a plastic substrate. The solvent of background electrolyte is evaporated from the open channel because of Joule heating during electrophoresis, leaving the dried electrophoretic bands to be directly analyzed by mass spectrometry via scanning electrostatic spray ionization. Proof-of-concept results are obtained with fluorescent dyes and antibiotics as the test samples, demonstrating an efficient on-chip detection platform based on the electrophoresis and electrostatic spray ionization mass spectrometry.

Electrostatic-Spray Ionization Mass Spectrometry

Natalia Gasilova, Hubert Girault, Yu Lu, Liang Qiao, Romain Sartor, Elena Tobolkina

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.

Amer Chemical Soc2012

Coupling Isoelectric Focusing Gel Electrophoresis to Mass Spectrometry by Electrostatic Spray Ionisation

Hubert Girault, Liang Qiao, Elena Tobolkina

Gel electrophoresis has been used for decades as a high-resolution separation technique for proteins and protein isomers, but limited in the coupling with MS because of low throughput and poor automaticity compared with LC-MS. In this work, we have developed an ambient ionisation strategy, electrostatic spray ionisation, for in-situ ionisation of proteins or peptides inside a surfactant-free polyacrylamide gel. The samples can be first separated by isoelectric focusing in a gel and then quickly in-situ detected by scanning the gel with the electrostatic spray ionisation mass spectrometry. With this strategy, nanograms of proteins or peptides inside a band are enough to be ionised for MS detection. This method for protein/peptide spots visualization is sensitive providing sample molecular weight information whilst avoiding spot staining and chemical extraction procedures that can introduce contaminants and sample loss. Proof-of-principle results have demonstrated that the electrostatic spray ionisation can produce sample ions from a complex background and with a spatial resolution matching the isoelectric focusing, therefore a good choice to couple directly isoelectric focusing gel electrophoresis with mass spectrometry.

American Chemical Society2013

Novel Analytical Methods for Allergy-Related Studies

Mikaël Frossard

Managing and treating diseases require a deep understanding of the pathologies coupled with accurate diagnostic procedures. The constant development and improvement of bioanalytical methods reflect the need of diagnosis ameliorations with respect to accuracy, sensitivity and efficiency. The field of allergy diagnosis is a good example. Despite affecting almost a third of the population in western countries, allergies still present complex challenges for the physicians in terms of diagnosis and allergen characterization. This thesis presents novel analytical methods developed for the fast and personalized allergy component-resolved diagnosis (CRD), the allergen identification and quantification in food matrices and the characterization of allergen modifications, notably nitration. Immunomagnetic separation (IMS) was used to perform the allergy diagnosis at the molecular level by extracting the IgE antibodies from allergic patient blood sera and probing them against individual allergens and natural allergenic extracts. The fast and highly selective IMS procedure was coupled to matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry (MS) to identify accurately and with high sensitivity the proteins recognized as sensitizers by the patient antibodies. The developed CRD methods were successful to diagnose cowâs milk, egg, peanut and tree nuts allergies, allowing the resolution of several complex clinical cases. Additionally, precious information was collected for the cross-reactivity and cross-sensitization in peanut and tree nuts allergies using blood sera from the PronNut clinical study. Allergen nitration is one of the potential causes for the increase in allergy prevalence observed recently and requires thorough investigation. Herein, various nitrating agents were tested and compared for in vitro allergen nitration. After modifying allergens using two of the most efficient nitrating procedures, the nitration sites were identified by bottom-up proteomics (BUP) and compared to the known epitopes, bringing useful information and novel insights to the problem of correlation between protein nitration and allergenicity. The presence of a minute amount of allergens in a food product is responsible for potential life-threatening reactions in highly allergic patients and sensitive analytical methods are therefore required in food quality assessments. A proteomic-based method was used for the quality control of several food products suspected of causing severe reactions for peanut and hazelnut-allergic patients. Peptide markers were identified in a first screening of the food extracts by BUP using a high-resolution MS instrument. A standard addition method was then used to quantify peanut and hazelnut proteins by BUP using an untargeted âshotgunâ approach.

EPFL2020

Novel Analytical Methods for Allergy-Related Studies

Mikaël Frossard

EPFL2020