title: Enrichment and detection of tyrosine-nitrated proteins.
abstract: Nitrotyrosine is a post-translationally modified amino acid with distinctly different properties than tyrosine or any other of the genetically encoded amino acids. Detecting proteins containing nitrotyrosine is the first step towards a better understanding of the role of nitrotyrosine in health and disease. Moreover, quantifying the extent of nitrotyrosine and determining its location in a protein forms the basis for a better understanding of the effect of tyrosine nitration on biological function. Described in this unit is a method to detect tyrosine-nitrated proteins in tissue sections and on western blots after creating a fluorescent complex between aminotyrosine, salicylaldehyde, and Al(3+). In addition, an approach is detailed for labeling aminotyrosine with biotin to enrich peptides from complex samples. Both methods require reduction of nitrotyrosine to aminotyrosine, which can be achieved with sodium dithionite or hemin plus dithiothreitol.
authors.affiliation: Pharmaceutical Gene Modulation, Groningen Research Institute of Pharmacy, University of Groningen, The Netherlands.
authors.forename: Frank
authors.initials: F
authors.lastname: Dekker
authors.affiliation: Analytical Biochemistry, Groningen Research Institute of Pharmacy, University of Groningen, The Netherlands.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Abello
authors.affiliation: Pharmaceutical Gene Modulation, Groningen Research Institute of Pharmacy, University of Groningen, The Netherlands.
authors.forename: Rosalina
authors.initials: R
authors.lastname: Wisastra
authors.affiliation: Analytical Biochemistry, Groningen Research Institute of Pharmacy, University of Groningen, The Netherlands.
authors.forename: Rainer
authors.initials: R
authors.lastname: Bischoff
mesh_terms: D000818:Animals; D001710:Biotin / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D007120:Immunochemistry / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011506:Proteins / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins; C002744:3-nitrotyrosine; D014443:Tyrosine; D001710:Biotin