Volume 566 of the series Methods in Molecular Biology pp 137-163


Mass Spectrometric Identification of In Vivo Nitrotyrosine Sites in the Human Pituitary Tumor Proteome

  • Xianquan ZhanAffiliated withCharles B. Stout Neuroscience Mass Spectrometry Laboratory, Department of Neurology, University of Tennessee Health Science Center Email author 
  • , Dominic M. DesiderioAffiliated withCharles B. Stout Neuroscience Mass Spectrometry Laboratory, Departments of Neurology and Molecular Sciences and the Cancer Institute, University of Tennessee Health Science Center

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The chemically stable tyrosine nitration of a protein involves the addition of a nitro group (–NO2) to the phenolic ring of a tyrosine residue, which may be associated with nervous system physiological and pathological processes. Identification of nitrotyrosine sites on a protein could clarify the functional significance of the modification. Due to the rarity of nitrotyrosine sites in a proteome, tandem mass spectrometry, coupled with different techniques that isolate and enrich nitrotyrosine-containing proteins from a pituitary proteome, is currently the most effective method for site identification. Commercially available nitrotyrosine polyclonal/monoclonal antibodies enable one to detect nitrotyrosine-containing proteins in a two-dimensional gel electrophoresis (2DGE) map, and to preferentially enrich nitrotyrosine-containing proteins with immunoprecipitation. Our present protocols have integrated different isolation/enrichment techniques (2DGE; Western blots; nitrotyrosine immunoaffinity precipitation) and two different tandem mass spectrometry methods (MALDI-MS/MS; ESI-MS/MS) to determine the amino acid sequence of nitrotyrosine-containing peptides that derive from nitrated proteins. Bioinformatics tools are then used to correlate nitrotyrosine sites with a functional domain/motif in order to understand the relationship between tyrosine nitration and the structural/functions of proteins.

Key words

Nitrotyrosine Nitroproteomics Two-dimensional gel electrophoresis Nitrotyrosine immunoaffinity enrichment Tandem mass spectrometry Bioinformatics