An Automated Comprehensive Ultrasensitive Two Dimensional Capillary Electrophoresis System For Proteomic Analysis

Author : Rafał Marszałek
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (817 download)

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Download or read book Towards Proteome-on-a-chip written by Rafał Marszałek and published by . This book was released on 2011 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Column-based separation techniques, such as liquid chromatography and capillary electrophoresis (CE), are often used to facilitate the hyphenation and sample transfer to a spectroscopic analysis stage due to their ability to be operated in a highly automatic and high-throughput style. Although there is some evidence in the literature for the use of capillary electrophoresis to deposit samples on a solid substrate for further analysis with mass spectrometry (MS) or other techniques, only a few examples of continuous deposition have so far been demonstrated. All of these, moreover, deposit sample at high substrate velocities, leading to the analyte zones spread over large distances and to decreased signal density in the subsequent analysis. Additionally, it is mainly MS that is used for sample analysis (with only one example of protein immunoblotting detection demonstrated). Recent developments in the field of infrared spectroscopy have, however, led to the invention of novel techniques, e.g. electron-vibration-vibration two-dimensional infrared spectroscopy (EVV 2DIR), which can be used for protein identification, quantification and other analysis. In this thesis a new workflow for top-down proteomic analysis (i.e. analysis of intact proteins) is proposed. In this strategy a number of mild separation steps - either fractionating the sample with size exclusion chromatography, sucrose gradients etc. or purification with affinity chromatography - are followed by capillary zone electrophoresis. CE is also used as an interface: the sample is slowly electrodeposited on the solid substrate. The speed of deposition ensures the high degree of analyte zone concentration on the substrate - leading to higher signal density and thus improving the sensitivity of any subsequent analysis. Finally, different optical read-out methods are employed for protein identification and quantitation, namely EVV 2DIR, modified Western blotting and imaging of the protein intrinsic (tryptophan) fluorescence. As the first step to establishing the workflow, an automated capillary electrophoresis instrument with a deposition interface was built and characterised in terms of its reproducibility. It was shown that an excellent migration time reproducibility of less than 0.5% can be achieved. Some inherent limitations of the interface are discussed in the thesis and the importance of the buffer system choice is emphasised. Additionally, a number of buffer systems were characterised in terms of their spectral and other properties to facilitate that choice. The interface platform was then applied to demonstrate feasibility of hyphenation between capillary electrophoresis and EVV 2DIR spectroscopy. In the proof-of-principle experiment five peptides were separated with CE, deposited on the solid substrate and subsequently imaged with intrinsic fluorescence read-out and EVV 2DIR. It was demonstrated that the peptide separations with the number of theoretical plates reaching 105 are attainable and the sample can be successfully deposited without loss of resolution. Due to the yet limited sensitivity of 2DIR read-out the final spectra of the deposited peptides were not obtained. As an alternative approach, coupling with the Western blotting read-out was suggested. Here, the chaperonin containing TCP-I was affinity purified, its subunits fractionated with CE and deposited on the nitrocellulose coated slides. The procedure was completed with the immunoblotting analysis. A proof-of-principle experiment was completed and the possible further improvements to the setup are discussed at length. 4.