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Title: Investigation of monolithic materials for protein sample preparation
Author: Alzahrani, Eman Saad
Awarding Body: University of Hull
Current Institution: University of Hull
Date of Award: 2012
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Proteomics plays an important role in the recognition of diseases and the understanding of biological processes. Sample preparation is a bottleneck in systems for chemical analysis and it is a required step in proteomics in order to remove interferences and preconcentrate the proteins. In addition, protein reduction and alkylation before digestion is a required step in proteomics to facilitate protein unfolding and increase the efficiency of enzymes in digesting proteins. The purpose of this study was to develop new techniques to address some of the shortcomings of current sample preparation methods, and provide short sample preparation time. Much research in recent years has focused on porous monolithic materials since they are highly permeable to liquid flow and show high mass transfer compared with common packed beds. This study has focused on the use of organic polymer- and inorganic silica-based monolithic materials for protein sample preparation. The organic polymer monolith used in this study was a butyl methacrylate-co-ethylene dimethacrylate (BuMA-co-EDMA) monolith that was fabricated inside the borosilicate tube using photoinitiated polymerisation. The porous properties of the fabricated monolith were controlled by adjusting the composition of the porogenic solvent in the polymerisation mixture. The results indicated that using MeOH/1-propanol as a porogenic solvent produced a polymer-based monolith with high surface area (56.89 m² g¯¹); however, it lacked the desired permeability and porosity when fabricated inside a glass microchip. Evaluation of its performance was carried out by extraction of four standard proteins that were insulin, cytochrome C, myoglobin, and hemoglobin and the extraction recovery was in the range (79.1-98.4 %). A monolithic silica rod was fabricated without cracks inside a heat shrinkable tube and then compared with the same material whose surface has been modified with octadecyl groups in order to use them for preconcentration/extraction of proteins. Their performance was evaluated using eight standard proteins, namely insulin, cytochrome C, lysozyme, myoglobin, β-lactoglobulin, ovalbumin, hemoglobin, and bovine serum albumin. The results show that recovery of the proteins was achieved by both columns with variable yields; however, the octadecylated silica monolith gave higher recoveries (92.7 - 109.7%) than the non-modified silica monolith (25.5 - 97.9%). This was followed by a new process for the fabrication of a silica-based monolith inside a glass microchip, which was successfully developed for use in microchip-based solid phase extraction of proteins. This was achieved by placement of the monolithic silica disk inside the extraction chamber in the base plate of the microchip, followed by thermal bonding of the two plates of the glass microchip at 575 °C for 3 hours. By doing this, the problem of shrinkage in the silica skeleton during preparation was avoided completely. The monolithic silica disk inside the glass microchip was subsequently modified with octadecyl groups for increased protein binding capacity. The performance of the microchip was evaluated using the extraction of standard proteins mixed with a high concentration of the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The results show that the octadecylated silica monolith was permeable, has the ability to remove impurities, and achieved a high extraction recovery of the proteins (94.8-99.7%) compared with conventional octadecylated silica particles (48.3-91.3%). The intra-batch and inter-batch RSDs were in the range of 2.0-4.5% and 2.9-6.4%, respectively. Comparison between the fabricated device and a commercial cartridge for the preconcentration of proteins in skimmed cows milk and hen egg white showed the ability of the device to successfully enrich protein mixtures from more realistic samples. This new microfluidic device for protein extraction may find an application in the area of proteomic research. A novel approach for immobilisation of the reducing reagent on the surface of the silica-based monolith in order to use it for protein reduction and alkylation was successfully developed. This was carried out by silanisation of the surface of the silica-based monolith with (3-aminopropyl)triethoxysilane (APTES), followed by immobilisation of the reducing reagent, tris (2-carboxyethyl) phosphine hydrochloride (TCEP) on the surface of the amino-bonded silica monolith. The fabricated monolith was characterised using IR spectroscopy, EDX analysis, BET model, and measuring the contact angle of deionised water. The fabricated monolith was evaluated for its use in protein reduction and alkylation in one single step at 60 °C by injection of a mixture consisting of 40 μL denatured protein and 60 μL iodoacetamide solution into the fabricated microchip, followed by using MALDI-TOF-MS instrument for qualitative confirmation. The results show that the fabricated microchip-based silica monolith has the ability to reduce and alkylate insulin in 30 min, and lysozyme in 45 min. Although this method was shown to require sample desalting to remove denaturant (urea) and the performance of the fabricated monolith had low intra-chip reproducibility, the method was simple, reduced the risk of contamination, decreased the number of processing steps, and results in lower amounts of the sample and reagents compared with the conventional techniques for proteomics sample preparation. More work is required to fully optimise this approach to protein sample preparation.
Supervisor: Welham, Kevin; McCreedy, Tom Sponsor: Taif University (Saudi Arabia)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Chemistry