Selective Affinity Enrichment of Nitrotyrosine-Containing Peptides for Quantitative Analysis in Complex Samples

Protein
tyrosine nitration by oxidative and nitrate stress is important
in the pathogenesis of many inflammatory or aging-related diseases.
Mass spectrometry analysis of protein nitrotyrosine is very challenging
because the non-nitrated peptides suppress the signals of the low-abundance
nitrotyrosine (NT) peptides. No validated methods for enrichment of
NT-peptides are currently available. Here we report an immunoaffinity
enrichment of NT-peptides for proteomics analysis. The effectiveness
of this approach was evaluated using nitrated protein standards and
whole-cell lysates in vitro. A total of 1881 NT sites were identified
from a nitrated whole-cell extract, indicating that this immunoaffinity-MS
method is a valid approach for the enrichment of NT-peptides, and
provides a significant advance for characterizing the nitrotyrosine
proteome. We noted that this method had higher affinity to peptides
with N-terminal nitrotyrosine relative to peptides with other nitrotyrosine
locations, which raises the need for future study to develop a pan-specific
nitrotyrosine antibody for unbiased, proteome-wide analysis of tyrosine
nitration. We applied this method to quantify the changes in protein
tyrosine nitration in mouse lungs after intranasal poly­(I:C) treatment
and quantified 237 NT sites. This result indicates that the immunoaffinity-MS
method can be used for quantitative analysis of protein nitrotyrosines
in complex samples.