Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.478937
Title: Toward a microfluidic system for proteomics
Author: Al Lawati, Haider A. J.
ISNI:       0000 0001 3406 8319
Awarding Body: University of Hull
Current Institution: University of Hull
Date of Award: 2007
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Abstract:
There has been much interest in the application of microfluidic systems to proteomics in recent years. This is because of the many unique advantages afforded by the reduced dimensions of microfluidic systems compared to classical methods. The reduction in the reaction vessel dimensions leads to a high degree of control, higher sensitivity, better selectivity and reduced analysis time. Initial experimental results have shown the possibility for developing a novel approach for proteomic analysis. A highly efficient protein digestion microdevice was fabricated using commercially available immobilized trypsin on agarose beads, packed into a silica capillary and connected directly to an electrospray mass spectrometer via a ‘microtight T’ connector, from which aqueous acetic acid (0.2 %) was pumped to the mass spectrometer ion source. Six proteins with molecular masses ranging from 2846 to 77703 Da were digested separately, each within eight minutes using this system. In a second set of experiments a short monolithic separation column was fabricated and placed after the immobilized trypsin capillary. This system demonstrated partial separation of the tryptic peptides generated, and detection limits in the pmol range were obtained by utilization of this separation column. The methodology was then transferred to a glass microchip and a novel system was fabricated. The system consists of an on-chip protein digestion channel and on-chip monolithic ion exchange separation column. The digestion system performance was evaluated using single proteins and mixtures of protein. Significant reduction in the digestion time was observed in comparison to the traditional digestion method and a complete digestion was obtained for cytochrome C in less than a minute while a protein mixture was digested within five minutes. The on-chip separation column was initially evaluated using the tryptic digest of cytochrome C. High column efficiency was obtained as indicated from the peak width at half height measurement data (varying between 19.0 to 24.9 s). A detection limit of 3.7 pmol was obtained; lower detection limits may be obtained if a nano-electrospray source were to be used. The separation column was also evaluated by separating a tryptic digest of a mixture of four proteins. Results showed at least 40 % improvement in the sequence coverage due to the separation achieved. In another experiment, the system was successfully used for protein digestion and a first dimension separation followed by an off-line second dimension separation. This was demonstrated using a tryptic digest of BSA and a number of additional peptides were identified as a result of the first dimension separation carried out using the microfluidic system. The system was also tested with a biological sample and initial results gave positive indications; however, this has to be confirmed after optimization of the protein extraction method. The developed system can be used along with the commercially available on-chip reverse phase columns to perform on-chip protein digestion and two dimensional separation of the generated peptides in a high throughput system for the analysis of biological samples. Another novel microfluidic system was also fabricated. The system consists of an on-chip protein digestion channel and on-chip monolithic reverse phase separation column. The microfluidic system was evaluated using cytochrome C, BSA and a mixture of four proteins. The separation and digestion were completed within one hour. However, the column performance was lower than that of the on-chip monolithic ion exchange column previously fabricated as indicated from the peak width at half height measurement data (varying between 30.0 to 115.0 s).
Supervisor: Welham, Kevin ; Watts, Paul Sponsor: Jami'at al-Sultan Qabus
Qualification Name: Thesis (Ph.D.) Qualification Level: Thesis or dissertation
EThOS ID: uk.bl.ethos.478937  DOI: Not available
Keywords: Chemistry
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