Abstract
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.
Highlights
Oxidative damage of proteins is involved in mechanisms such as insulin resistance, cell-cycle arrest, senescence, and apoptosis.[1]Reactive oxygen/nitrogen species (ROS/RNS) such as the superoxide radical (O2−) converted to peroxynitrite and nitric oxide (NO) can be (ONOOH/ONOO−),[2] which is a very strong oxidant
The results shown in this paper follow a doubly resonant setup, where the IR beams are in resonance with coupled vibrational modes of the molecule, whereas the ωγ beam is scattered from a nonresonant electronic level
We report on the ability of EVV 2DIR spectroscopy to identify tyrosine nitration in model peptides, to quantify nitration levels in peptide mixtures down to 1%, and to differentiate between nTyr residues containing a neutral versus deprotonated hydroxyl group
Summary
Oxidative damage of proteins is involved in mechanisms such as insulin resistance, cell-cycle arrest, senescence, and apoptosis.[1]Reactive oxygen/nitrogen species (ROS/RNS) such as the superoxide radical (O2−) converted to peroxynitrite and nitric oxide (NO) can be (ONOOH/ONOO−),[2] which is a very strong oxidant. Oxidative damage of proteins is involved in mechanisms such as insulin resistance, cell-cycle arrest, senescence, and apoptosis.[1]. The principal targets of peroxynitrite in proteins are sulfhydryl and tyrosyl side chains with nitration of tyrosine (Tyr) to 3-nitrotyrosine (nTyr) being one of the most extensively studied ROS/RNS-driven post-translational modifications (PTMs).[3−5]. Protein tyrosine nitration is thought to interfere with a range of biochemical processes[6] and is known to prevent PTMs such as phosphorylation.[5,7] Accumulation of nTyr is seen in neurodegenerative diseases,[8−10] such as amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, and Parkinson’s disease, as well as in chronic inflammation[5] and cardiovascular disease.[11,12]. Tyr residue (Tyr327) has been shown to have a positive regulatory effect through the activation of p53,13 a tumorsuppressant protein that acts as a transcription factor for various proteins involved in apoptosis and cell cycle arrest.
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