Chemical approach for specific enrichment and mass analysis of nitrated peptides

The analysis and detection of 3-nitrotyrosine are biologically and clinically important because protein tyrosine nitration is known to be involved in a number of biological phenomena such as cellular signal transduction, pathogenesis of inflammatory responses, and age-related disorders. However, the main obstacles in the study are low abundance of nitrated species and lack of efficient enrichment methods. Here in, we suggest a new chemical approach to analyze nitrated peptides using mass spectrometry by incorporating specific tagging groups in the peptides through simple chemical transformations. Nitro groups on tyrosine side chains of nitrated peptides were subjected to reduction to give rise to amine which was further converted to metal-chelating motif. Mass analyses verified that Ni(2+)-NTA magnetic agarose beads selectively captured and isolated the modified peptides, i.e., nitrated peptides, by strong and specific metal chelating interactions. We further demonstrated the utility of our approach by detection of nitrated peptides in complex samples such as tryptic peptide mixtures of bovine serum albumin (BSA) and a HeLa cell lysate.