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

Hubert Girault, Liang Qiao, Elena Tobolkina

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.

American Chemical Society2013

On the utility of predictive chromatography to complement mass spectrometry based intact protein identification

Hisham Ben Hamidane, Yury Tsybin

The amino acid sequence determines the individual protein three-dimensional structure and its functioning in an organism. Therefore, "reading" a protein sequence and determining its changes due to mutations or post-translational modifications is one of the objectives of proteomic experiments. The commonly utilized approach is gradient high-performance liquid chromatography (HPLC) in combination with tandem mass spectrometry. While serving as a way to simplify the protein mixture, the liquid chromatography may be an additional analytical tool providing complementary information about the protein structure. Previous attempts to develop "predictive" HPLC for large biomacromolecules were limited by empirically derived equations based purely on the adsorption mechanisms of the retention and applicable to relatively small polypeptide molecules. A mechanism of the large biomacromolecule retention in reversed-phase gradient HPLC was described recently in thermodynamics terms by the analytical model of liquid chromatography at critical conditions (BioLCCC). In this work, we applied the BioLCCC model to predict retention of the intact proteins as well as their large proteolytic peptides separated under different HPLC conditions. The specific aim of these proof-of-principle studies was to demonstrate the feasibility of using "predictive" HPLC as a complementary tool to support the analysis of identified intact proteins in top-down, middle-down, and/or targeted selected reaction monitoring (SRM)-based proteomic experiments.

Springer-Verlag2012

Analysis of Intact Monoclonal Antibody IgG1 by Electron Transfer Dissociation Orbitrap FTMS

Luca Fornelli, Yury Tsybin

The primary structural information of proteins employed as biotherapeutics is essential if one wishes to understand their structure-function relationship, as well as in the rational design of new therapeutics and for quality control. Given both the large size (around 150 kDa) and the structural complexity of intact immunoglobulin G (IgG), which includes a variable number of disulfide bridges, its extensive fragmentation and subsequent sequence determination by means of tandem mass spectrometry (MS) are challenging. Here, we applied electron transfer dissociation (ETD), implemented on a hybrid Orbitrap Fourier transform mass spectrometer (FTMS), to analyze a commercial recombinant IgG in a liquid chromatography (LC)-tandem mass spectrometry (MS/MS) top-down experiment. The lack of sensitivity typically observed during the top-down MS of large proteins was addressed by averaging time-domain transients recorded in different LC-MS/MS experiments before performing Fourier transform signal processing. The results demonstrate that an improved signal-to-noise ratio, along with the higher resolution and mass accuracy provided by Orbitrap FTMS (relative to previous applications of top-down ETD-based proteomics on IgG), is essential for comprehensive analysis. Specifically, ETD on Orbitrap FTMS produced about 33% sequence coverage of an intact IgG, signifying an almost 2-fold increase in IgG sequence coverage relative to prior ETD-based analysis of intact monoclonal antibodies of a similar subclass. These results suggest the potential application of the developed methodology to other classes of large proteins and biomolecules. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.019620, 1758-1767, 2012.

Amer Soc Biochemistry Molecular Biology Inc2012