5 years ago

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

Selective Affinity Enrichment of Nitrotyrosine-Containing Peptides for Quantitative Analysis in Complex Samples
Rosario Maroto, Yueqing Zhang, Yingxin Zhao, Allan R. Brasier, Hong Sun
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.

Publisher URL: http://dx.doi.org/10.1021/acs.jproteome.7b00275

DOI: 10.1021/acs.jproteome.7b00275

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