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
