Mass Spectrometric Identification of In Vivo Nitrotyrosine Sites in the Human Pituitary Tumor Proteome
- 8 June 2009
- book chapter
- Published by Springer Science and Business Media LLC in Methods in Molecular Biology
- Vol. 566, 137-163
- https://doi.org/10.1007/978-1-59745-562-6_10
Abstract
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.Keywords
This publication has 21 references indexed in Scilit:
- Linear ion-trap mass spectrometric characterization of human pituitary nitrotyrosine-containing proteinsInternational Journal of Mass Spectrometry, 2007
- Nitroproteins from a human pituitary adenoma tissue discovered with a nitrotyrosine affinity column and tandem mass spectrometryAnalytical Biochemistry, 2006
- The human pituitary nitroproteome: detection of nitrotyrosyl-proteins with two-dimensional Western blotting, and amino acid sequence determination with mass spectrometryBiochemical and Biophysical Research Communications, 2004
- Differences in the spatial and quantitative reproducibility between two second-dimensional gel electrophoresis systemsElectrophoresis, 2003
- Spot volume vs. amount of protein loaded onto a gel: A detailed, statistical comparison of two gel electrophoresis systemsElectrophoresis, 2003
- A reference map of a human pituitary adenoma proteomeProteomics, 2003
- Investigation of tyrosine nitration in proteins by mass spectrometryJournal of Mass Spectrometry, 2001
- Analysis of peptides and proteins containing nitrotyrosine by matrix-assisted laser desorption/ionization mass spectrometryJournal of the American Society for Mass Spectrometry, 2001
- Intercellular Communication in the Anterior PituitaryEndocrine Reviews, 2000
- Nitric oxide synthase in the rat anterior pituitary gland and the role of nitric oxide in regulation of luteinizing hormone secretion.Proceedings of the National Academy of Sciences, 1993