Mass spectrometric identification of in vivo nitrotyrosine sites in the human pituitary tumor proteome.
The chemically stable tyrosine nitration of a protein involves the addition of a nitro group (-NO(2)) 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.
Zhan
Xianquan
X
Department of Neurology, University of Tennessee Health Science Center, Memphis, TN, USA. xqzhan2001@yahoo.com
Desiderio
Dominic M
DM
D019295:Computational Biology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods*; D010911:Pituitary Neoplasms / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry
D011506:Proteins; C002744:3-nitrotyrosine; D014443:Tyrosine