Detection of antimicrobial resistance-associated proteins by titanium dioxide-facilitated intact bacteria mass spectrometry

Titanium dioxide-modified target plates were developed to enhance intact bacteria analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The plates were designed to photocatalytically destroy the bacterial envelope structure and improve the ionization efficiency of intracellular components, thereby promoting the measurable mass range and the achievable detection sensitivity. Accordingly, a method for rapid detection of antimicrobial resistance-associated proteins, conferring bacterial resistance against antimicrobial drugs, was established by mass spectrometric fingerprinting of intact bacteria without the need of any sample pre-treatment. With this method, the variations in resistance proteins expression levels within bacteria were quickly measured from the relative peak intensities. This approach of resistance proteins detection directly from intact bacteria by mass spectrometry is useful for fast discrimination of antimicrobial-resistant bacteria from their non-resistant counterparts whilst performing species identification. Also, It could be used as a rapid and convenient way for initial determination of the underlying resistance mechanisms.

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

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

EPFL2003

Mass Spectrometry based Analytical Methods for Proteome Analysis and Redoxomics

Liang Qiao

EPFL2013