Identification and relative quantification of tyrosine nitration in a model peptide using two-dimensional infrared spectroscopy

Nitration of tyrosine in proteins and peptides is a post-translational modification that occurs under conditions of oxidative stress. It is implicated in a variety of medical conditions, including neurodegenerative and cardiovascular diseases. However, monitoring tyrosine nitration and understanding its role in modifying biological function remains a major challenge. In this work, we investigate the use of electron-vibration-vibration (EVV) two-dimensional infrared (2DIR) spectroscopy for the study of tyrosine nitration in model peptides. We demonstrate the ability of EVV 2DIR spectroscopy to differentiate between the neutral and deprotonated states of 3-nitrotyrosine, and we characterize their spectral signatures using information obtained from quantum chemistry calculations and simulated EVV 2DIR spectra. To test the sensitivity of the technique, we use mixed-peptide samples containing various levels of tyrosine nitration, and we use mass spectrometry to independently verify the level of nitration. We conclude that EVV 2DIR spectroscopy is able to provide detailed spectroscopic information on peptide side-chain modifications and to detect nitration levels down to 1%. We further propose that lower nitration levels could be detected by introducing a resonant Raman probe step to increase the detection sensitivity of EVV 2DIR spectroscopy. (Graph Presented)

Coherent multidimensional infrared spectroscopy – application to the study of biomolecules under oxidative stress.

There is a growing body of evidence which suggests post-translational modifications occurring under oxidative stress (oxPTMs) play an important role in both human health and disease. The focus of the work described in this thesis has been on the use of coherent multidimensional spectroscopy (CMDS) to perform detection and quantification of oxPTMs in a label-free and non-destructive manner. Electron-Vibration-Vibration (EVV) two-dimensional infrared (2DIR) spectroscopy is a CMDS technique which is able to directly observe intra- and intermolecular interactions. As a result, EVV 2DIR spectroscopy is particularly useful for characterising (oxPTMs). EVV 2DIR spectroscopy employs one near-IR and two mid-IR picosecond excitation beams to probe vibrational couplings in a sample via a four-wave mixing process. This results in the spread of vibrational coupling information across two dimensions, which leads to spectral decongestion and the ability to directly analyse vibrational modes within complex molecules, such as proteins. Here, tyrosine (Tyr) nitration is used as a study model due to its importance in inflammatory diseases, amongst other pathologies. Results are presented for various nitration models and will demonstrate EVV 2DIR spectroscopy's ability to identify, relatively quantify and characterise the effect of nitration of tyrosine side-chains.Open Acces