Abstract
Biologic nitration of protein tyrosine (to form 3-nitrotyrosine) is a recently described phenomenon that is associated with many diseases. We have devised a proteomic methodology to identify these modified proteins. This utilizes protein fractionation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), partial transfer onto polyvinylidene difluoride (PVDF) membranes, and Western blot analysis using an antinitrotyrosine antibody to identify the proteins. Alignment of the Western blots with the partially transferred 2-D PAGE gels enables identification of immunopositive protein spots. These are then excised and trypsin digested. Proteins are then identified using either matrix-assisted laser desorption ionization-time of flight mass spectrometry or capillary liquid chromatography tandem electrospray mass spectrometry. Nonspecific crossreactivity of the antibodies is determined using reduction of protein bound 3-nitrotyrosine to 3-amino tyrosine using sodium dithionite.
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Aulak, K.S., Koeck, T., Crabb, J.W., Stuehr, D.J. (2004). Proteomic Method for Identification of Tyrosine-Nitrated Proteins. In: Hassid, A. (eds) Nitric Oxide Protocols. Methods in Molecular Biology™, vol 279. Humana Press. https://doi.org/10.1385/1-59259-807-2:151
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DOI: https://doi.org/10.1385/1-59259-807-2:151
Publisher Name: Humana Press
Print ISBN: 978-1-58829-237-7
Online ISBN: 978-1-59259-807-6
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