Capillary Electrophoresis with Multiple Readout Techniques for Protein Analysis
Author(s)
Goyder, Miriam Sarah
Type
Thesis
Abstract
In the era of proteomics, new technologies in separation and identification
are required. Separation methods, such as capillary electrophoresis or liquid
chromatography, are a crucial part of high throughput proteomic workflows.
In this thesis, novel approaches to proteomics using capillary electrophoresis
are presented.
A platform of technologies based on capillary electrophoresis with continuous
deposition of separated proteins onto metallic substrates enables subsequent
analyses and identification. Since sample deposition and identification are decoupled, multiple readout techniques can be explored. Readout
techniques used include matrix assisted laser desorption/ionisation mass
spectrometry (MALDI-MS), electron-vibration-vibration two dimensional infrared
spectroscopy (EVV 2DIR) and fluorescence microscopy. This technology
was used without the deposition interface, to achieve advances in ribosomal
separations or with the deposition interface, to develop new proteomic
strategies of separation and readout.
The eukaryotic ribosomal proteins were separated using capillary electrophoresis
for the first time. Over 26 peaks were resolved in less than
10 minutes. An outstanding RSD migration time of < 0.5% was achieved,
demonstrating that the readout could provide a ribosomal ' fingerprint'.
Separations of proteins were successfully analysed using a standard MALDIMS
instrument. This work was advanced by the offline coupling of CE to
MALDI-imaging and applied to the ribosomal proteins to demonstrate a
novel workflow from cell culture to protein identification.
Quantitative analysis of protein levels is an important part of proteomics,
but is difficult to achieve using mainstream technologies with high throughput
and accuracy. EVV 2DIR is a non-linear spectroscopy which is able to achieve
absolute quantification of proteins.[1] Coupling of EVV 2DIR to CE (CE-
2DIR) was demonstrated through the deposition and analysis of peptide and
proteins. CE-2DIR offers great promise as a new proteomic tool.
are required. Separation methods, such as capillary electrophoresis or liquid
chromatography, are a crucial part of high throughput proteomic workflows.
In this thesis, novel approaches to proteomics using capillary electrophoresis
are presented.
A platform of technologies based on capillary electrophoresis with continuous
deposition of separated proteins onto metallic substrates enables subsequent
analyses and identification. Since sample deposition and identification are decoupled, multiple readout techniques can be explored. Readout
techniques used include matrix assisted laser desorption/ionisation mass
spectrometry (MALDI-MS), electron-vibration-vibration two dimensional infrared
spectroscopy (EVV 2DIR) and fluorescence microscopy. This technology
was used without the deposition interface, to achieve advances in ribosomal
separations or with the deposition interface, to develop new proteomic
strategies of separation and readout.
The eukaryotic ribosomal proteins were separated using capillary electrophoresis
for the first time. Over 26 peaks were resolved in less than
10 minutes. An outstanding RSD migration time of < 0.5% was achieved,
demonstrating that the readout could provide a ribosomal ' fingerprint'.
Separations of proteins were successfully analysed using a standard MALDIMS
instrument. This work was advanced by the offline coupling of CE to
MALDI-imaging and applied to the ribosomal proteins to demonstrate a
novel workflow from cell culture to protein identification.
Quantitative analysis of protein levels is an important part of proteomics,
but is difficult to achieve using mainstream technologies with high throughput
and accuracy. EVV 2DIR is a non-linear spectroscopy which is able to achieve
absolute quantification of proteins.[1] Coupling of EVV 2DIR to CE (CE-
2DIR) was demonstrated through the deposition and analysis of peptide and
proteins. CE-2DIR offers great promise as a new proteomic tool.
Date Issued
2011-09
Date Awarded
2011-10
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
deMello, Andrew
Ces, Oscar
Klug, David
Creator
Goyder, Miriam Sarah
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)