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A Method for Analysis of Nitrotyrosine-Containing Proteins by Immunoblotting Coupled with Mass Spectrometry

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Mitochondrial Medicine

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2276))

Abstract

Nitrotyrosine formation is caused by presence of reactive oxygen and nitrogen species. Nitration is a very selective process leading to specific modification of only a few tyrosines in protein molecule. 2D electrophoresis and western blotting techniques coupled with mass spectrometry are common methods used in analysis of proteome. Here we describe protocol for analysis of peroxynitrite-induced protein nitration in isolated mitochondria. Mitochondrial proteins are separated by 2D electrophoresis and transferred to nitrocellulose membrane. Membranes are then incubated with antibodies against nitrotyrosine. Positive spots are compared with corresponding Coomassie-stained gels, and protein nitration is confirmed with mass spectrometry techniques.

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Acknowledgments

This work was supported by grant of GACR No. P303/11/0298 and GACR No. 15-01948S.

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Correspondence to Matej Kohutiar .

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Kohutiar, M., Eckhardt, A. (2021). A Method for Analysis of Nitrotyrosine-Containing Proteins by Immunoblotting Coupled with Mass Spectrometry. In: Weissig, V., Edeas, M. (eds) Mitochondrial Medicine . Methods in Molecular Biology, vol 2276. Springer, New York, NY. https://doi.org/10.1007/978-1-0716-1266-8_28

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  • DOI: https://doi.org/10.1007/978-1-0716-1266-8_28

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  • Publisher Name: Springer, New York, NY

  • Print ISBN: 978-1-0716-1265-1

  • Online ISBN: 978-1-0716-1266-8

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