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 AnalysisReport as inadecuate




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 - Download this document for free, or read online. Document in PDF available to download.

1

Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, 4202 E. Fowler Ave., Tampa, FL 33620, USA

2

Training Institute, Thermo Fisher Scientific, 1400 Northpoint Parkway, Ste 10., West Palm Beach, FL 33407, USA



Current address: Center for Drug Discovery and Innovation, Proteomics Facility, University of South Florida, 3720 Spectrum Blvd., Tampa, FL 33612, USA





*

Author to whom correspondence should be addressed.



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. View Full-Text

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





Author: Kent W. Seeley 1,†, Alison R. Fertig 1, Craig P. Dufresne 2, Joao P. C. Pinho 1 and Stanley M. Stevens Jr. 1,*

Source: http://mdpi.com/



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