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, 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.

3-Nitrotyrosine quantification methods: Current concepts and future challenges

“…The abundance of protein or Tyr residues cannot predict whether they will be the target of PTN. Furthermore, not all Tyr residues in a protein are available for nitration, which may depend on their accessibility to the solvent [10,15]. For instance, although the human serum albumin (HSA), a protein most abundantly found in plasma, contains 18 Tyr residues, an in vitro study showed that only two of its Tyr residues are predominantly susceptible to nitration [14,16].…”

“…Several physiological and pathological conditions have been associated with increased nitration of proteins [15,21]. Table 1 shows the 3-NT concentration range usually found in both healthy and pathological states, as determined by different methods (discussed in detail in the following sections) and biological samples.…”

Pros and Cons of Current Approaches for Detecting Peroxynitrite and Their Applications

“…[53] Moreover, efforts are also made for developing various methods to identify nitration protein and nitrotyrosine site. [54,55] With the development of novel techniques for special assessment of not only ONOO − but also nitration protein and nitrotyrosine sites, we will be able to better understand the oxidation and redox mechanisms in both physiological and pathological conditions. The progress offers new opportunities and platforms for drug discovery targeting peroxynitrite and other free radicals for therapeutic purposes.…”

NTyroSite: Computational Identification of Protein Nitrotyrosine Sites Using Sequence Evolutionary Features

“…Accurate identification of nitrotyrosine sites is an essential foundation for revealing the mechanism and function of nitroproteins [ 13 , 14 ]. To identify nitrotyrosine sites, various large-scale proteomic studies are widely adopted in numerous organisms, including Bacillus licheniformis , Bacillus subtilis , Bos taurus , Bungarus multicinctus , Capsella bursa-pastoris , Carica papaya , Cricetulus migratorius , Enterobacteria phage fd , Escherichia phage lambda , Enterobacteria phage T4 , Enterococcus faecalis , Escherichia coli , Fusarium oxysporum , Halobacterium salinarum , Hirudo medicinalis , Homo sapiens , Mus musculus , Naja atra , Naja melanoleuca , Neurospora crassa , Ophiophagus hannah , Oryctolagus cuniculus , Ovis aries , Finegoldia magna , Phascolopsis gouldii , Physeter catodon , Pseudomonas putida , Rattus norvegicus , Rhodococcus rhodochrous , Saccharomyces cerevisiae , Schizophyllum commune , Staphylococcus aureus , Streptomyces albogriseolus , Struthio camelus , Sus scrofa , Thermus thermophilus , Tobacco mosaic virus , and Protobothrops flavoviridis [ 1 , 7 , 8 , 10 , 14 , 15 ].…”

“…To identify nitrotyrosine sites, various large-scale proteomic studies are widely adopted in numerous organisms, including Bacillus licheniformis , Bacillus subtilis , Bos taurus , Bungarus multicinctus , Capsella bursa-pastoris , Carica papaya , Cricetulus migratorius , Enterobacteria phage fd , Escherichia phage lambda , Enterobacteria phage T4 , Enterococcus faecalis , Escherichia coli , Fusarium oxysporum , Halobacterium salinarum , Hirudo medicinalis , Homo sapiens , Mus musculus , Naja atra , Naja melanoleuca , Neurospora crassa , Ophiophagus hannah , Oryctolagus cuniculus , Ovis aries , Finegoldia magna , Phascolopsis gouldii , Physeter catodon , Pseudomonas putida , Rattus norvegicus , Rhodococcus rhodochrous , Saccharomyces cerevisiae , Schizophyllum commune , Staphylococcus aureus , Streptomyces albogriseolus , Struthio camelus , Sus scrofa , Thermus thermophilus , Tobacco mosaic virus , and Protobothrops flavoviridis [ 1 , 7 , 8 , 10 , 14 , 15 ]. Despite the increasing number of experimentally identified nitrotyrosine sites, the mechanism of site-specific nitration modifications on tyrosine still remains largely unknown, possibly because of the constraints of measurement technologies [ 3 , 12 , 13 ]. On the other hand, experimental verification of nitrotyrosine sites is time-consuming, labor-intensive, and sometimes biased toward the abundant proteins.…”