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Int. J. Mol. Sci. 2014, 15(4), 6265-6285; doi:10.3390/ijms15046265
Article

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

1,†
, 1
, 2
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 and 1,*
Received: 30 January 2014; in revised form: 22 March 2014 / Accepted: 24 March 2014 / Published: 14 April 2014
(This article belongs to the Special Issue Fourier Transform Mass Spectrometry in Molecular Sciences)
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Abstract: 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.
Keywords: nitration; oxidative stress; immonium ion; high resolution mass spectrometry; liquid chromatography (LC)-MS/MS; FTMS nitration; oxidative stress; immonium ion; high resolution mass spectrometry; liquid chromatography (LC)-MS/MS; FTMS
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MDPI and ACS Style

Seeley, K.W.; Fertig, A.R.; Dufresne, C.P.; Pinho, J.P.C.; Jr., S.M.S. 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. Int. J. Mol. Sci. 2014, 15, 6265-6285.

AMA Style

Seeley KW, Fertig AR, Dufresne CP, Pinho JPC, Jr. SMS. 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. International Journal of Molecular Sciences. 2014; 15(4):6265-6285.

Chicago/Turabian Style

Seeley, Kent W.; Fertig, Alison R.; Dufresne, Craig P.; Pinho, Joao P.C.; Jr., Stanley M.S. 2014. "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." Int. J. Mol. Sci. 15, no. 4: 6265-6285.


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