Current Published Papers

Swann, M.J., Freeman, N.J., Carrington, S., Ronan, G., Barrett, P., – Quantifying Structural Changes and Stoichiometry of Protein Interactions Using Size and Density Profiling. Letters in Peptide Science, (2003), 10 487-494.

Alternative reference

Swann, M., Freeman,N., Carrington,S., Ronan,G., Barrett, P., Quantifying structural changes and stoichiometry of protein interactions using size and density profiling. International Journal of Peptide Research and Therapeutics, (2003),10 (5-6) 487-494.

Koopmann , J. O., Blackburn , J., High Affinity Capture Surface for Matrix-Assisted Laser Desorption/Ionisation Compatible Protein Microarrays. Rapid Commun. Mass Spectrom., (2003), 17 455-462.

Abstract: A surface for the capture of biotin-tagged proteins on matrix-assisted laser desorption/ionization (MALDI) targets has been investigated. Binding of a poly-L-lysine poly(ethylene glycol)-biotin polymer to glass and gold surfaces has been demonstrated using dual wavelength interferometry. Biotinylated proteins were captured onto this surface using tetrameric neutravidin as a multivalent bridging molecule. Biotin tagging of proteins was achieved by chemical biotinylation or by expressing a protein with a biotinylation consensus sequence in E. coli. The specificity of the surface for biotin-tagged proteins allowed the purification of biotin-tagged glutathione-S-transferase from a bacterial lysate directly onto a MALDI target. Subsequently, the protein was digested on the MALDI target and a protein fingerprint analysis confirmed its presence directly, but no E. coli proteins were detected. Therefore, we conclude that this surface is highly specific for the capture of biotin-labelled proteins and has low non-specific binding properties for non-biotinylated proteins. Furthermore, protein– protein interactions using biotinylated lectins were investigated, and the selective capture of the glycoprotein fetuin with wheat germ agglutinin was demonstrated. Also, immobilised Arachis hypogea agglutinin recognised a minor asialo component of this glycoprotein on the array. The high affinity immobilisation of proteins onto this surface allowed effective desalting procedures to be used which improved the desorption of high molecular weight proteins. Another aspect of this surface is that a highly ordered coupling of the analyte can be achieved which eliminates the search for the sweet spot and allows the creation of densely packed protein microarrays for use in mass spectrometry.

Cross, G.H., Brand, A. S., Popplewell, J. F., Peel, L. L., Swann, M.J., Freeman N. J., A New Quantitative Optical Biosensor for Protein Characterisation. Biosensors and Bioelectronics, (2003), 19 383-390.

Keywords: Optical biosensor; Protein characterisation; X-ray crystallography

Abstract: A new optical biosensor is described based on a dual waveguide interferometric technique. By addressing the waveguide structure with alternate polarisations the optogeometrical properties (density and thickness) of adsorbed protein layers at the sensor (solid)-liquid interface have been determined. Differences in the waveguide mode dispersion between the transverse electric (TE) and transverse magnetic (TM) modes allow unique solutions for adlayer thickness and refractive index to be determined at all stages during the formation process. The technique has been verified using standard protein systems and by comparing the data with published work using X-ray crystallography and neutron reflection techniques. The data obtained was found to be in excellent agreement with previously reported X-ray experiments given that typical film thicknesses for streptavidin layers were in the range 5.5_ 6.5 nm compared with the short axis crystal structure of between 4.8 and 5.6 nm. The precision of the measurements taken was of the order of 40 pm with respect to adsorbed adlayer thicknesses. This biosensor approach provides measurements of both thickness and density of adlayers to a high precision, simultaneously and in real time enabling detail of the structure and function of proteins to be elucidated. From such data it is possible to obtain information on the orientation, distortion and efficiency of immobilisation procedures as well as the interaction event of interest. The technique is expected to find utility with those interested in protein structure and function. This is an area of growing importance within the life sciences as the demand for quantitative analytical techniques increases with the growth in ‘proteomics’.