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

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