New analytical tools combining gel electrophoresis and mass spectrometry

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

Coupling Isoelectric Focusing Gel Electrophoresis to Mass Spectrometry by Electrostatic Spray Ionisation

Gel electrophoresis has been used for decades as a high-resolution separation technique for proteins and protein isomers, but limited in the coupling with MS because of low throughput and poor automaticity compared with LC-MS. In this work, we have developed an ambient ionisation strategy, electrostatic spray ionisation, for in-situ ionisation of proteins or peptides inside a surfactant-free polyacrylamide gel. The samples can be first separated by isoelectric focusing in a gel and then quickly in-situ detected by scanning the gel with the electrostatic spray ionisation mass spectrometry. With this strategy, nanograms of proteins or peptides inside a band are enough to be ionised for MS detection. This method for protein/peptide spots visualization is sensitive providing sample molecular weight information whilst avoiding spot staining and chemical extraction procedures that can introduce contaminants and sample loss. Proof-of-principle results have demonstrated that the electrostatic spray ionisation can produce sample ions from a complex background and with a spatial resolution matching the isoelectric focusing, therefore a good choice to couple directly isoelectric focusing gel electrophoresis with mass spectrometry

Standard addition strip for quantitative electrostatic spray ionization mass spectrometry analysis: Determination of caffeine in drinks

Standard addition strips were prepared for the quantitative determination of caffeine in different beverages by electrostatic spray ionisation mass spectrometry (ESTASI-MS). The gist of this approach is to dry spots of caffeine solutions with different concentrations on a polymer strip, then to deposit a drop of sample mixed with an internal standard, here theobromine on each spot and to measure the mass spectrometry signals of caffeine and theobromine by ESTASI-MS. This strip approach is very convenient and provides quantitative analyses as accurate as the classical standard addition method by MS or liquid chromatography

Electrostatic-spray ionization mass spectrometry sniffing for perfume fingerprinting

RATIONALE: The perfume market is growing significantly, and it is easy to find imitative fragrances of probably all types of perfume. Such imitative fragrances are usually of lower quality than the authentic ones, creating a possible threat for perfume companies. Therefore, it is important to develop efficient chemical analysis techniques to screen rapidly perfume samples. METHODS: Electrostatic-spray ionization (ESTASI) was used to analyze directly samples sprayed or deposited on different types of paper. A linear ion trap mass spectrometer was used to detect the ions produced by ESTASI with a modified extended transfer capillary for ’sniffing’ ions from the paper. RESULTS: Several commercial perfumes and a model perfume were analyzed by ESTASI-sniffing. The results obtained by paper ESTASI-MS of commercial fragrances were compared with those obtained from ESI-MS. In addition, a commercial fragrance was first nebulized on the hand and then soaked up by blotting paper, which was afterwards placed on an insulating plate for ESTASI-MS analysis. Analysis of peptides and proteins was also performed to show that the paper ESTASI-MS could be used for samples with very different molecular masses. CONCLUSIONS: Paper ESTASI-MS yields a rapid fingerprinting characterization of perfume fragrances, avoiding timeconsuming sample-preparation steps, and thereby performing a rapid screening in a few seconds

Proteins/peptides purification by a three-well OFFGEL electrophoresis with immobilized ultra narrow pH gradient gels

Purification and desalting of protein and peptide samples by a three-well OFFGEL electrophoresis with immobilized ultra narrow pH gradient gels is proposed as a fast preparative strategy for proteomics. The gist of this strategy is to separate the proteins and peptides according to their isoelectric point and to isolate those of a given pI value equal to the mean pH value of the gel. The present approach has been demonstrated both on protein mixtures and a digested Escherichia coli protein extract. UV-Vis spectroscopy, MALDI-MS, SDS-PAGE and LC-MS/MS were employed for the quantitative and qualitative characterization of the separation results. The electrophoretic methodology has been simulated by finite element methods

Segmented Field OFFGEL® Electrophoresis

A multielectrode setup for protein OFFGEL electrophoresis that significantly improves protein separation efficiency has been developed. Here, the electric field is applied by segments between seven electrodes connected in series to six independent power supplies. The aim of this strategy is to distribute evenly the electric field along the multiwell system, and as a consequence to enhance electrophoresis in terms of separation time, resolution, and protein collection efficiency, while minimizing the overall potential difference and therefore the Joule heating. The performances were compared to a standard two-electrode setup for OFFGEL fractionation of a protein mixture, using UV-Vis spectroscopy for quantification and MALDI-MS for identification. The electrophoretic separation process was simulated, and optimized by solving the time-dependent Nernst–Planck differential equation

Electrostatic-Spray Ionization Mass Spectrometry

An electrostatic-spray ionization (ESTASI) method has been used for mass spectrometry (MS) analysis of samples deposited in or on an insulating substrate. The ionization is induced by a capacitive coupling between an electrode and the sample. In practice, a metallic electrode is placed close to but not in direct contact with the sample. Upon application of a high voltage pulse to the electrode, an electrostatic charging of the sample occurs leading to a bipolar spray pulse. When the voltage is positive, the bipolar spray pulse consists first of cations and then of anions. This method has been applied to a wide range of geometries to emit ions from samples in a silica capillary, in a disposable pipet tip, in a polymer microchannel, or from samples deposited as droplets on a polymer plate. Fractions from capillary electrophoresis were collected on a polymer plate for ESTASI MS analysis

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

Electrostatic Spray Ionization Mass Spectrometry Imaging

Imaging samples on a surface by mass spectrometry (MS) requires the combination of MS detection with a scanning mode that enables localized desorption and ionization and/or detection of sample analytes with good spatial resolution. We have developed a new mass spectrometry imaging (MSI) method based on electrostatic spray ionization. It works under ambient conditions and can be applied to a wide range of molecules providing quantitative MS analysis even in the presence of salts in excess. 2D MS images of protein and peptide spots, inkjet-printed black dye patterns, and cells were obtained. The presented novel ambient ionization mass spectrometry imaging method can find many applications in analytical and bioanalytical chemistr