Novel Analytical Methods for Allergy-Related Studies

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.

Multiplexed Middle-Down Mass Spectrometry as a Method for Revealing Light and Heavy Chain Connectivity in a Monoclonal Antibody

Daniel Ayoub, Luca Fornelli, Natalia Gasilova, Anton Kozhinov, Laure Menin, Konstantin Nagornov, Kristina Srzentic, Yury Tsybin

Pairing light and heavy chains in monoclonal antibodies (mAbs) using top-down (TD) or middle-down (MD) mass spectrometry (MS) may complement the sequence information on single chains provided by high-throughput genomic sequencing and bottom-up proteomics, favoring the rational selection of drug candidates. The 50 kDa F(ab) subunits of mAbs are the smallest structural units that contain the required information on chain pairing. These subunits can be enzymatically produced from whole mAbs and interrogated in their intact form by TD/MD MS approaches. However, the high structural complexity of F(ab) subunits requires increased sensitivity of the modern TD/MD MS for a comprehensive structural analysis. To address this and similar challenges, we developed and applied a multiplexed TD/MD MS workflow based on spectral averaging of tandem mass spectra (MS/MS) across multiple liquid chromatography (LC)-MS/MS runs acquired in reduced or full profile mode using an Orbitrap Fourier transform mass spectrometer (FTMS). We first benchmark the workflow using myoglobin as a reference protein, and then validate it for the analysis of the 50 kDa F(ab) subunit of a therapeutic mAb, trastuzumab. Obtained results confirm the envisioned benefits in terms of increased signal-to-noise ratio of product ions from utilizing multiple LC-MS/MS runs for TD/MD protein analysis using mass spectral averaging. The workflow performance is compared with the earlier introduced multiplexed TD/MD MS workflow based on transient averaging in Orbitrap FTMS. For the latter, we also report on enabling absorption mode FT processing and demonstrate its comparable performance to the enhanced FT (eFT) spectral representation.

AMER CHEMICAL SOC2018

Miniaturized analytical systems for mass spectrometry-based protein studies

Mélanie Abonnenc

Current proteomic strategies depend strongly on the development of analytical methodologies and instrumentation. In parallel to the development of mass spectrometry (MS) - based proteomic workflows, microfluidic devices emerged in this field as a flexible tool for rapid and sensitive protein studies. In this context, the present work focuses on the development of miniaturized analytical systems for protein studies, especially by electrospray ionization mass spectrometric detection. Several approaches have been proposed to complement the mass spectrometric analysis of tryptic peptides using chemical tagging to isolate specific subclasses of proteins by affinity baits or for quantitation purposes. Optimization of a chemical tagging reaction in a sandwich mixer-reactor that consists of mixing two solutions providing the reactants in a channel, one central laminar flow being sandwiched between two outer flow solutions, was first investigated by finite-element method. The numerical simulations have highlighted the importance of positioning the reactant presenting the lowest diffusion coefficient close to the sidewall, as well as decreasing the outer flow velocities, to enhance a chemical reaction because of the parabolic flow profile across the microchannel. This optimization is even more relevant for consecutive reactions. As infusion-based electrospray microchips are known to present great advantages in terms of sensitivity compared to standard ionization source, an ESI emitter microchip based on polymer photo-ablation was designed. First, the hyphenation of the chip in a LC-MS workflow was successfully achieved and evaluated by comparison with a standard pneumatically-assisted ESI source. To perform enhanced on-chip post-column derivatization of peptides, a substitute design to the sandwich mixer-reactor was explored to improve reaction in a microchip before ESI – MS analysis. The electrospray micromixer chip includes a passive mixing unit to perturbate the flow along the microchannel. The mixing efficiency was demonstrated by fluorescence imaging, and on-chip chemical derivatization of peptides and kinetic studies before mass spectrometry were achieved. The on-line LC-MS derivatization of cysteinyl peptides was shown to provide a more confident and accurate protein identification by adding information on the peptidic sequence. Within the development of electrospray microchips for mass spectrometry, many efforts have been put forward in the hyphenation of functional units. Immobilization of functionalized stationary phase in microfluidic systems is widely used to achieve protein or peptide isolation, sample cleaning, separation or reaction. In this context, magnetic beads have proven to be quite useful compared to other methods, such as immobilized packed beads or monoliths, because of their high and reversible magnetization, and various surface functionalization. A polymer microchip including a magnetic track array was designed to focus the magnetic field generated by permanent magnets towards precise locations along the microchannel. A multi-plug magnetic bead capture was obtained and a significant increase of bead capture efficiency was demonstrated both numerically and experimentally, which is beneficial for affinity separation applications, as example. The research in microfluidic front-end devices for mass spectrometry is actually oriented to the development of multi-spray interfaces for high-throughput analysis. An electrospray microchip with a multi-track array was developed. The capability of the device to screen alternatively up to six samples and to perform relative quantification was assessed. Soft ionization techniques have revolutionized the analysis of proteins by mass spectrometry. In contrast with the major content of the thesis dealing with microfluidic systems for protein analysis, the last chapter presents a novel ambient ionization method, so-called membrane-desorption electrospray ionization (M-DESI). The principle is similar to the desorption electrospray ionization (DESI) method. But, in the M-DESI approach the sample is desorbed directly from a mesh membrane positioned vertically and in-axis between the ESI emitter and the mass spectrometer inlet. Analysis of peptides and proteins was demonstrated as a proof-of-concept, offering great perspectives for detection after separation on membrane.

EPFL2009

Mass Spectrometry Based Analysis and Bioanalysis

Hongyan Bi

MS (mass spectrometry) has been developed as an important analytical tool to study large molecules including polymer macromolecules and proteins. Its instrumentation technique including ionization methods, mass analyzer and detector systems has been greatly improved from last century to present. Bio-molecules and organic macromolecules tend to be fragile and fragment when they are ionized by conventional ionization methods. ESI (electrospray ionization) and MALDI (matrix assisted laser desorption/ionization) are two relatively tender ionization methods and suitable for the ionization of big molecules. The study on mechanism surround these two ionization methods is a hot issue. For MALDI, one wants to figure out what exactly happens when the laser irradiates the analyte, where in-plume reaction occurs. As an example, the photo-triggered reaction can induce the fragmentation of peptides or proteins. Proteolysis is a key step for proteomic study especially bottom-up proteomic strategy. Porous materials have been developed in last decades. The catalysis of porous materials to enzymatic reaction is a hot issue, and has been demonstrated in much published works. But the mechanism of how different porous materials affect the reaction is not completely clear yet. We compared several different porous material influences for the reaction efficiencies. A kinetic model was proposed to explain and foresee the proteolysis. The model was verified by integrated CE (capillary electrophoresis)/MS detection system, where a custom-designed CE-MS interface was utilized to collect the fraction of digests for off-line MALDI TOF (time-of-flight) MS analysis. From an instrumental point, we then introduce Al-foil as disposable and once-used MALDI-target substitute, by which the sample preparation for MALDI TOF MS analysis is greatly simplified. The regeneration procedure of typical target in each run and attrition of target-plate can be neglected after holding this Al-foil on a commercially available target plate. This substrate provides comparable detection limits (LOD) of protein and digests (peptides) as the typical stainless steel MALDI target plate. The pliability of Al-foil makes it easy to be printed. While nano-scale semiconductor materials such as TiO2 have been widely used for dye-sensitized solar cell study. Following the MALDI target holder study, TiO2 nanoparticles were prepared as optimized ink and fabricated on the Al-foil via screen- and rotogravure- printing procedures. The woven mesh and engraved image carrier can be designed and controlled for different print pattern. The given apparatus here can provide two different TiO2 functionalized Al-foil. The functionalized foil then was then taken for the analysis of matrix free LDI (laser desorption ionization) and the study of protein phosphorylation, which is one of the most common and important protein post-translational modifications that occur in animal cells. Since the typical organic matrix interference always makes noise signals in low mass/charge on a mass spectrum, especially below 1000 or 700 Th, matrix free LDI is introduced to study the low mass molecules or small molecules. In this context, besides TiO2 nanoparticles, we also investigate CdSe semiconductor quantum dots, and electrochemically polymerized film of an aromatic diamine for the LDI analysis. A standard peptide was applied for the feasibility study of LDI based on these substrates. Semiconductor quantum dots are also hot target molecules for the dye-sensitized solar cell. By comparing the photocurrents on different quantum dots fabricated electrodes, the electron transfer process inside was simulated to understand the kinetics, which is also expected to guide the LDI study including the mechanism involved.

EPFL2010