Evaluation of a Method for Nitrotyrosine Site Identification and Relative Quantitation Using a Stable Isotope-Labeled Nitrated Spike-In Standard and High Resolution Fourier Transform MS and MS/MS Analysis
Angiotensin I, Animals, Cattle, Cell Line, Chromatography, High Pressure Liquid, Fourier Analysis, Isotope Labeling, Mass Spectrometry, Nitrogen Isotopes, Peroxynitrous Acid, Rats, Serum Albumin, Bovine, Tandem Mass Spectrometry, Tyrosine
Digital Object Identifier (DOI)
The overproduction of reactive oxygen and nitrogen species (ROS and RNS) can have deleterious effects in the cell, including structural and possible activity-altering modifications to proteins. Peroxynitrite is one such RNS that can result in a specific protein modification, nitration of tyrosine residues to form nitrotyrosine, and to date, the identification of nitrotyrosine sites in proteins continues to be a major analytical challenge. We have developed a method by which 15N-labeled nitrotyrosine groups are generated on peptide or protein standards using stable isotope-labeled peroxynitrite (O15NOO−), and the resulting standard is mixed with representative samples in which nitrotyrosine formation is to be measured by mass spectrometry (MS). Nitropeptide MS/MS spectra are filtered using high mass accuracy Fourier transform MS (FTMS) detection of the nitrotyrosine immonium ion. Given that the nitropeptide pair is co-isolated for MS/MS fragmentation, the nitrotyrosine immonium ions (at m/z = 181 or 182) can be used for relative quantitation with negligible isotopic interference at a mass resolution of greater than 50,000 (FWHM, full width at half-maximum). Furthermore, the standard potentially allows for the increased signal of nitrotyrosine-containing peptides, thus facilitating selection for MS/MS in a data-dependent mode of acquisition. We have evaluated the methodology in terms of nitrotyrosine site identification and relative quantitation using nitrated peptide and protein standards
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Citation / Publisher Attribution
International Journal of Molecular Sciences, v. 15, issue 4, p. 6265-6285
Scholar Commons Citation
Seeley, Kent W.; Fertig, Alison R.; Dufresne, Craig P.; Pinho, Joao P. C.; and Stevens, Stanley M. Jr., "Evaluation of a Method for Nitrotyrosine Site Identification and Relative Quantitation Using a Stable Isotope-Labeled Nitrated Spike-In Standard and High Resolution Fourier Transform MS and MS/MS Analysis" (2014). Cell Biology, Microbiology, and Molecular Biology Faculty Publications. 11.