Mass spectrometric investigation of protein nitration in inflammation and neurodegeneration

Abstract: The nitration of tyrosine seems to be very common in human diseases and at least forty different diseases have been reported to be associated with an increased nitration of proteins. The nitration of proteins results in a modification of tyrosine, 3-nitrotyrosine (3-NT), which is stable. This modification is believed to cause toxic effects to the cell and has e.g. been shown to cause increased degradation of proteins and decreased activity of enzymes. The principal methods for investigation of this type of protein modification are assays based on antibody-based techniques, e.g. immunohistochemistry. The majority of all investigations have used such methods. However, these methods often lack necessary information for interpretation of the validity of the results. In this work we describe how 3-NT can be measured in biological samples with an assay using gas chromatography and mass spectrometry. The assay is based on the reduction of the nitrotyrosine in the samples to aminotyrosine and subsequent derivatization to a di-O-methyl-di-N-heptafluorobutyryl derivative. The method was shown to have a limit of detection of 0.03 nM and a limit of quantification of 0.3 nM in human plasma. The method was used to measure free 3-NT and nitration of proteins in a model of zymosan-induced peritonitis. The results from these investigation show increases of 3-NT in serum from day one, together with increases of protein nitration in the cerebral cortex at day eight after the zymosan treatment. However, when applied to CSF samples from healthy controls and patients with either amyotrophic lateral sclerosis or Alzheimer s disease, no obvious difference between the groups could be seen.