Bottomley, Anna Louise; (2003) The action of peroxynitrite on proteins. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Oxidative damage to proteins can occur under physiological conditions through the action of reactive oxygen species, including those containing nitrogen such as peroxynitrite. Peroxynitrite has been shown in vitro to target tyrosine and tryptophan residues in proteins through free radical addition to produce 3-nitrotyrosine and nitrotryptophan. In this work, we show that mass spectral patterns associated with 3-nitrotyrosine and nitrotryptophan containing peptides allow identification of peptides containing these modifications. Quadrupole time-of-flight mass spectrometry (Q-TOF MS) was used to characterise several peptides containing 3-nitrotyrosine and one containing nitrotryptophan, derived from albumin and transferrin treated with either peroxynitrite or 3-morpholino-sydnonimine (SIN-1). A unique series of modified peptides were also found for the reacted proteins that did not relate to the nitration of tyrosine residues. Sequencing of these peptides found that nitration is not the only modification that tyrosine can undergo. Tyrosine was found to also undergo dinitration, oxidation and dimerisation. It was also found that other amino acids underwent modification when exposed to peroxynitrite or SIN-1. Histidine showed modifications of + 126 Da and + 252 Da, lysine + 28 Da, cysteine + 25 Da and threonine - 18 Da. Several proteins containing 3-nitrotyrosine were isolated from the cerebral spinal fluid of acute lymphoblastic patients using western blotting with the antibody antinitrotyrosine. One of these proteins was identified as albumin using matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS). These observations suggest that Q-TOF MS/MS can provide a selective method for the analysis and characterisation of amino acids modified by peroxynitrite and that a combination of western blotting and MALDI-TOF MS can be utilised to isolate and identify proteins containing 3-nitrotyrosine. However, it does highlight that caution is required when attributing observed changes to protein activity, after exposure to peroxynitrite, to only the nitration of tyrosine residues.

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