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

Yingxin Zhao, Yueqing Zhang, Hong Sun, Rosario Maroto, Allan R. Brasier

Research output: Contribution to journalArticlepeer-review

20 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)2983-2992
Number of pages10
JournalJournal of Proteome Research
Issue number8
StatePublished - Aug 4 2017


  • affinity enrichment
  • mass spectrometry
  • posttranslational modifications
  • protein tyrosine nitration

ASJC Scopus subject areas

  • General Chemistry
  • Biochemistry


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