Two-dimensional infrared spectroscopy for protein analysis

A number of forms of coherent multi-dimensional vibrational spectroscopy (CMDVS) have been identified as being useful for addressing a range of biological problems. Here a particular member of this family of spectroscopies, electronvibration- vibration two-dimensional infrared (EVV 2DIR) spectroscopy (also known as DOubly-Vibrationally Enhanced InfraRed (DOVE-IR)), is explored for its possible utility for two particular bioanalytical applications; protein identification and the study of enzyme mechanisms. The main focus of this work is on the development of EVV 2DIR as a tool for high-throughput, label-free proteomics, in particular for protein identification and absolute quantification. The protein fingerprinting strategy is based on the identification of proteins through their spectroscopically determined amino acid compositions. To this end, spectral signatures of amino acid side chains (tyrosine, phenylalanine and tryptophan) have been identified, as well as those from CH2 and CH3 groups which have been found to be appropriate for use as internal references. The intensities of these cross peaks are measured to give proteins’ amino acid compositions in the form of amino acid / CHx ratios. Specialised databases comprising the amino acid / CHx ratios of proteins have been developed for achieving protein identifications using the EVV 2DIR data. The second strand of this research considers the potential of triply resonant EVV 2DIR for studying protein structures and mechanisms. It is possible to use the electronic polarising properties of EVV 2DIR to good effect to achieve significant enhancement of the signal size when probing a chromophore. Here this effect is demonstrated for the case of bacteriorhodopsin (bR) membranes isolated from Halobacterium salinarium. The signal enhancement that is achievable from the retinal chromophore at the heart of bR makes it possible to study this whilst avoiding the surrounding protein