New ionization and tagging methods for mass spectrometry

Mass spectrometry (MS) is an essential detection tool in bioanalytical chemistry owing to its exceptional selectivity and sensitivity, paired with rapid analyte identification and quantification. In this thesis, two classical MS and a newly developed ionization method are employed, including matrix-assisted laser desorption/ionization (MALDI) MS, electrospray ionization (ESI) MS and electrostatic spray ionization (ESTASI) MS. To meet the emerging challenges in bioanalytical chemistry, from disease diagnosis to drug development, new MS-based analytical methods ought to be developed with respect to high sensitivity, throughput, speed, as well as sample consumption and experimental simplification. For these purposes, this thesis presents four analytical strategies combined with MS detection for improved analytical performance in different research fields. Two classical analytical tools, thin layer chromatography (TLC) and 384-well plate, were coupled with ESTASI-MS respectively. In ESTASI-TLC-MS, ESTASI was applied to extract and identify a wide range of molecules of different polarities and chemical structures from both hydrophilic or hydrophobic silica plates with high sensitivity and minimal sample consumption in the femtomole range. In 384-well plate ESTASI-MS, the commercial 384-well plate could work as a container and an emitter for sample spray ionization, without any liquid delivery system or any additional interface. This approach provides fast and high throughput analyses for the large batches of reactions, such as enzyme assay and drug metabolism. Tyrosinase-catalyzed tyrosine oxidation in the presence or absence of inhibitors and cytochrome P450-catalyzed metabolic reactions of two drugs were studied respectively. To improve analysis sensitivity, two strategies, mass barcoded gold nanoparticles (Mb-AuNPs) for MALDI-MS signal amplification and microfabricated on-chip spyhole (Ø 10-12 ¿m) emitter for nanoelectrospray (spyhole-nanoESI), were developed, leading to a low sample consumption and high analysis efficiency. Mb-AuNPs combined with magnetic separation were applied for multiplex cow¿s milk allergy diagnosis in a component-resolved manner. IgE antibodies (Abs) could be extracted from a patient¿s blood serum by the formation of a sandwich structure between allergenic proteins-coated Mb-AuNPs and anti-human IgE Abs-functionalized magnetic beads (MBs). Detection of Mb-AuNPs by MALDI-MS provides a limit of detection (LOD) down to picograms per milliliter level for specific IgE Abs from only 1 ¿L of patient¿s blood serum. To take advantages of microfluidics in low sample consumption and easy integration, a novel interface of spyhole-nanoESI was designed for coupling microfluidics with MS and showed an improved sensitivity than standard ESI. This disposable microchip coupled with MS/MS shows potential application in cancer diagnosis by the successful detection of a small cell lung cancer biomarker in 1 ¿L of human serum at the extensive stage, without any complicated sample preparation steps.

From allergen detection to protein-ligand interactions

Natalia Gasilova

Fundamental biological and biochemical studies, proteomics, practical nutrition-related and medical applications, all the research fields, where bioanalytical chemistry is an essential tool, constantly require the improvement of existing techniques of bioanalysis together with the development of new ones. To successfully meet and solve emerging scientific challenges, modern bioanalytical methodologies should provide high sensitivity, selectivity and efficiency of the analysis in combination with low sample consumption, fast experimental procedure and high throughput. This thesis presents various methods and tools that have been developed to address different aspects of bioanalytical chemistry starting from allergen detection in hypoallergenic alimentary products for food safety control and finishing with protein-ligand interaction studies. In all techniques described below, mass spectrometry (MS) was employed as a detection method owing to its exceptional selectivity and sensitivity, paired with rapid analyte identification. Immunoaffinity capillary electrophoresis (IACE) using magnetic beads as immunosupport was coupled with matrix-assisted laser desorption/ionization (MALDI) MS to perform sensitive analysis of two major bovine milk allergens, beta-lactoglobulin and alpha-lactalbumin, for food safety and quality control. Then, the developed technique was extended to the performance of an effective component-resolved diagnostic of cow`s milk allergy using a tiny amount of patient blood serum, while offering the possibility of unusual allergen discovery and allergenomic studies. As sample pretreatment is the key step of all analytical procedures, a new on-chip technique named solid-phase extraction-gradient elution mass spectrometry (SPE-GEMS) was demonstrated for the effective sample purification, enrichment and fractionation followed by on-line electrospray ionization (ESI) MS detection. In this method, especially useful for proteomic studies, magnetic beads coated with reverse-phase chromatographic coating were used as SPE sorbent for the analysis of various peptide mixtures, providing an advantage of simple sorbent regeneration in a disposable manner. As a continuation of this project, described SPE-GEMS procedure was modified for processing more complex biological samples with their identification by tandem MS detection and, hence, was referred to as SPE-GEMS/MS. Due to the application of mesoporous silica shell magnetic core microspheres as a stationary phase, the new technique displayed high selectivity and sensitivity towards large hydrophobic peptides, which is a valuable characteristic for middle-down proteomic experiments. To ensure fast and high throughput experimental performance together with low sample consumption, the analytical procedure can be designed using droplet-based microfluidics. Therefore, a new way of coupling electrostatic-spray ionization (ESTASI) MS with a water-in-oil droplet system was realized via microchip with a spyhole. The developed system provided efficient analysis of the droplet content, without dilution or an additional oil removal step. It was successfully applied for the performance of single-phase reactions, as well as biphasic reactions, namely protein-ligand interaction between beta-lactoglobulin and acetate of liposoluble vitamin E.

EPFL2014

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

Polymer nanospray interfaces for on-line electrochemically induced modifications

Tatiana Rohner

The aim of this work has been to design, and fabricate a miniaturized electrospray-type interface for mass spectrometry analysis, which allows the nebulization of solutions by applying a high potential difference between the source and the mass spectrometer. The fabrication of the nanospray emitter is based on UV-laser photoablation of polymer substrates. Thick-film channel flow electrodes can be integrated in the microfluidic system, allowing the application of a high voltage to generate electrospray from the open outlet of the microchannel. The polymer-based interface first presented is characterized by a flat edge interface. However, a V-shaped nozzle has been found to present higher stability and ease of use. This design also allows the generation of spray by applying high voltage through an electrode located in a solution reservoir, thereby inducing an electroosmotic pumping. Taking advantage of the intrinsic electrolytic behavior of electrospray, a specific electrochemically induced tagging of free cysteine residues located in proteins can be performed on-line. A p-hydroquinone buffer is used to perform a continuous infusion of protein solution. When in contact with the high-voltage microelectrode, p-hydroquinone undergoes oxidation, thereby producing protonated p-benzoquinone. The latter reacts with free cysteine residues, following a Michael-type addition. The modification of the protein can be followed thanks to MS analyses. A complete study of the mechanisms involved in the electrochemically induced tagging with hydroquinone has been carried out by cyclic voltammetry and digital simulation. The process of the tagging consists of an ECE mechanism, since the formed adduct is stabilized in the reduced state, and is thus free to be oxidized. The rate constant of the chemical reaction has been extracted, by performing cyclic voltammetry studies and modeling the whole system by digital simulations. Finite element simulations have also permitted to highlight several mechanistic aspects of the electrotagging. The nanospray interface has been modeled as a channel-flow electrode cell. After validation of the model with respect to the experimental results, kinetics and mass transport conditions have been investigated. The flow profile, as well as the kinetic rate constant, has been shown to be of great importance in the electrochemically induced tagging. The use of a sacrificial electrode as metallic ion source to generate electrospray has also been investigated. Transition metal electrodes, i.e. copper, zinc, nickel and iron, as well as silver, were used to get on-line complexation with model peptides. It has been demonstrated that the use of in-reservoir sacrificial electrode was an efficient method to generate metal ions in order to form complexes with peptides, without the addition of metallic salts. These studies have demonstrated that polymer nanospray interfaces can be advantageously used as analytical devices, together with their primary function, i.e. ionization sources for protein analyses by mass spectrometry.

EPFL2003