Biological and Biomedical Applications of Two-Dimensional Vibrational Spectroscopy: Proteomics, Imaging, and Structural Analysis

Abstract

In the last 10 years, several forms of two-dimensional infrared (2DIR) spectroscopy have been developed, such as IR pump-probe spectroscopy and photon-echo techniques. In this Account, we describe a doubly vibrationally enhanced four-wave mixing method, in which a third-order nonlinear signal is generated from the interaction of two independently tunable IR beams and an electron-polarizing visible beam at 790 nm. When the IR beams are independently in resonance with coupled vibrational transitions, the signal is enhanced and cross-peaks appear in the spectrum. This method is known as either DOVE (doubly vibrationally enhanced) four-wave mixing or EVV (electron-vibration-vibration) 2DIR spectroscopy. We begin by discussing the basis and properties of EVV 2DIR. We then discuss several biological and potential biomedical applications. These include protein identification and quantification, as well as the potential of this label-free spectroscopy for protein and peptide structural analysis. In proteomics, we also show how post-translational modifications in peptides (tyrosine phosphorylation) can be detected by EVV 2DIR spectroscopy. The feasibility of EVV 2DIR spectroscopy for tissue imaging is also evaluated. Preliminary results were obtained on a mouse kidney histological section that was stained with hematoxylin (a small organic molecule). We obtained images by setting the IR frequencies to a specific cross-peak (the strongest for hematoxylin was obtained from its analysis in isolation; a general CH(3) cross-peak for proteins was also used) and then spatially mapping as a function of the beam position relative to the sample. Protein and hematoxylin distribution in the tissue were measured and show differential contrast, which can be entirely explained by the different tissue structures and their functions. The possibility of triply resonant EVV 2DIR spectroscopy was investigated on the retinal chromophore at the centre of the photosynthetic protein bacteriorhodopsin (bR). By putting the visible third beam in resonance with an electronic transition, we were able to enhance the signal and increase the sensitivity of the method by several orders of magnitude. This increase in sensitivity is of great importance for biological applications, in which the number of proteins, metabolites, or drug molecules to be detected is low (typically pico- to femtomoles). Finally, we present theoretical investigations for using EVV 2DIR spectroscopy as a structural analysis tool for inter- and intramolecular interaction geometries.