On-Chip Spyhole Nanoelectrospray Ionization Mass Spectrometry for Sensitive Biomarker Detection in Small Volumes

A polyimide microfluidic chip with a microhole emitter (Ø 10–12 μm) created on top of a microchannel by scanning laser ablation has been designed for nanoelectrospray ionization (spyhole-nanoESI) to couple microfluidics with mass spectrometry. The spyhole-nanoESI showed higher sensitivity compared to standard ESI and microESI from the end of the microchannel. The limits of detection (LOD) for peptide with the spyhole-nanoESI MS reached 50 pM, which was 600 times lower than that with standard ESI. The present microchip emitter allows the analysis of small volumes of samples. As an example, a small cell lung cancer biomarker, neuron-specific enolase (NSE), was detected by monitoring the transition of its unique peptide with the spyhole-nanoESI MS/MS. NSE at 0.2 nM could be well identified with a signal to noise ratio (S/N) of 50, and thereby its LOD was estimated to be 12 pM. The potential application of the spyhole-nanoESI MS/MS in cancer diagnosis was further demonstrated with the successful detection of 2 nMNSE from1 μL of human serum. Before the detection, the serumsample spiked with NSE was first depleted with immune spin column, then desalted by centrifugal filter device, and finally digested by trypsin, without any other complicated preparation steps. The concentration matched the real condition of clinical samples. In addition, the microchips can be disposable to avoid any cross contamination. The present technique provides a highly efficient way to couple microfluidics with MS, which brings additional values to various microfluidics and MS-based analysis.

Mass Spectrometry based Analytical Methods for Proteome Analysis and Redoxomics

Liang Qiao

Proteomics is nowadays one of the most important research topics in life science for understanding many life activities from the molecular level. The objective is to find out proteins or modifications on proteins that regulate biological events, aging, diseases and so forth. Because of the complexity in the structure of proteins, the dynamic range of protein-abundances, and the differences from time-to-time and tissue-to-tissue, proteomics requires development of analytical methods with respect to resolution, sensitivity, speed, etc. In this thesis, analytical methods based on mass spectrometry are developed for proteome research and especially redox proteomics. Two major types of mass spectrometers are employed, including matrix-assisted laser desorption/ionization mass spectrometer (MALDI-MS) and electrospray ionization mass spectrometer (ESI-MS). Nano and micro metal oxide materials are used to modify the MALDI sample plate for in-situ extraction of phosphorylated peptides or for in-source photochemical reactions. The former is valuable for the study of protein posttranslational modifications. The latter results in online tagging of cysteine for counting the number of cysteine in peptides that is valuable information for the identification of cysteine-containing proteins. Functional microfluidic chips are designed as microreactor and ESI emitter for online chemical reactions followed with MS characterization. Specifically, the multi-channel microchip is applied in tyrosine nitration study. Tyrosine nitration is considered as a biomarker for radical associated diseases, such as cardiovascular, Alzheimer’s, and Parkinson’s diseases. With the microchip ESI-MS, a new pathway for tyrosine nitration induced by copper(II) is demonstrated. Considering that abnormally high concentration of copper(II) was found in the brain tissue of patient with Alzheimer’s disease, the current finding is important for understanding the mechanism of neurodegenerative diseases. An electrostatic-spray ionization (ESTASI) method is developed to generate ions from sample solutions. The ESTASI can be realized with various geometries, such as samples in a microchannel, in a capillary, in a micropipette and on a plastic plate. Comparing to ESI, voltage is not directly applied on the sample during ESTASI, thereby avoiding redox reactions during ionization. The ESTASI has been used to couple separation strategies with MS, including capillary electrophoresis and gel isoelectric focusing. These new methods can be useful in top-down proteomics and mass imaging.

EPFL2013

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

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