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.