Electrostatic spray ionization from 384-well microtiter plates for mass spectrometry analysis based enzyme assay and drug metabolism screening

We have realized the direct ionization of samples from wells of microtiter plates under atmospheric conditions for mass spectrometry analysis without any liquid delivery system or any additional interface. The microtiter plate is a commercially available 384-well plate without any modification, working as a container and an emitter for electrostatic spray ionization of analytes. The approach provides high throughput for the large batches of reactions and both the qualitative and quantitative analysis of a single compound or mixture. The limits of detection in small drug molecules, peptides and proteins are similar in comparison with standard direct infusion electrospray ionization. The analysis time per well is only seconds. These analytical merits benefit many microtiter plate-based studies such as combinatorial chemistry and high throughput screening in enzyme assay or drug metabolism. Herein, we illustrate the application in enzyme assay using tyrosine oxidation catalyzed by tyrosinase in the presence or absence of inhibitors. The potential application in drug development is also demonstrated with cytochrome P450-catalyzed metabolic reactions of two drugs in microtiter plates followed with direct ESTASI-MS/MS based characterization of the metabolism products.

New analytical tools combining gel electrophoresis and mass spectrometry

Elena Tobolkina

Proteomics has been one of the main projects challenging biological and analytical chemists for many years. The separation, identification and quantification of all the proteins expressed within biological systems remain the main objectives of proteomics. Due to sample complexity, the development of fractionation, separation, purification and detection techniques that possess appropriate resolution to separate a large number of proteins, as well as being sensitive and fast enough for high throughput protein analysis are required. The objective of this thesis was to develop new separation strategies for protein/peptide fractionation using gel electrophoresis and its further detection by mass spectrometry analysis. A multi-electrode set up based on OFFGEL electrophoresis was developed. The objective was to provide a more efficient application of the electric field for fast and improved protein separation. The results showed that using a multi-electrode setup provides not only a higher protein separation but also better protein collection efficiency. Electrophoretic separation using an ultra narrow pH gradient (UNPG) gel was adapted for a three-well OFFGEL device for fast sample purification and desalting. Purification of an E.coli extract was applied to demonstrate that electrophoretic separation with UNPG gels provides an efficient strategy for fast purification of complex biological samples and can be utilized as a preparative technique in proteomics. UNPG gels were also used to separate charge molecules taking place in a new electro-elution device. The molecules were washed from the gel surface by an aqueous buffer and collected for further analysis by mass spectrometry. The electroelution device provides a fast approach that avoids time-consuming steps of extraction from the polyacrylamide gel. An electrostatic spray ionization (ESTASI) mass spectrometry technique was developed in our group to ionize sample solutions on different substrates. ESTASI has been here coupled to isoelectric focusing and demonstrated to be a powerful tool, which can improve the detection sensitivity compared to standard visualizing protocols. Paper-ESTASI-MS was developed and applied for rapid perfume analysis of six authentic fragrances as a high throughput and sample preparation-free method. ESTASI was also applied for quantitative analysis of caffeine in different beverages using a standard addition strip. The results were compared to classical standard addition methods for MS or LC. It was shown that the strip strategy could be utilized for fast and accurate food analysis and quality control. Desalting and direct analysis of samples from ZipTip by ESTASI-MS has been demonstrated.

EPFL2014

New ionization and tagging methods for mass spectrometry

Xiaoqin Zhong

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.

EPFL2017

New ionization and tagging methods for mass spectrometry

Xiaoqin Zhong

EPFL2017

New analytical tools combining gel electrophoresis and mass spectrometry

Elena Tobolkina

EPFL2014