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

Kent W. Seeley¹  Alison R. Fertig¹  Craig P. Dufresne²  Joao P. C. Pinho¹ 

[1] Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, 4202 E. Fowler Ave., Tampa, FL 33620, USA; E-Mails:;Training Institute, Thermo Fisher Scientific, 1400 Northpoint Parkway, Ste 10., West Palm Beach, FL 33407, USA; E-Mail:

关键词: nitration;    oxidative stress;    immonium ion;    high resolution mass spectrometry;    liquid chromatography (LC)-MS/MS;    FTMS;   

DOI  :  10.3390/ijms15046265

来源: mdpi

【 摘 要 】

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.

【 授权许可】

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