Analysis of nitrated proteins and tryptic peptides by HPLC-chip-MS/MS: site-specific quantification, nitration degree, and reactivity of tyrosine residues.

The reaction products and pathways of protein nitration were studied with bovine serum albumin (BSA) and ovalbumin (OVA) nitrated by liquid tetranitromethane (TNM) or by gaseous nitrogen dioxide and ozone (NO(2)+O(3)). Native and nitrated proteins were enzymatically digested with trypsin, and the tryptic peptides were analyzed by high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) using a chip cube nano-flow system (Agilent). Upon nitration by TNM, up to ten of 17 tyrosine residues in BSA and up to five of ten tyrosine residues in OVA could be detected in nitrated form. Upon nitration by NO(2)+O(3), only three nitrated tyrosine residues were found in BSA. The nitration degrees of individual nitrotyrosine residues (ND(Y)) were determined by site-specific quantification and compared to the total protein nitration degrees (ND) determined by photometric detection of HPLC-DAD. The slopes of the observed linear correlations between ND(Y) and ND varied in the range of ~0.02-2.4 for BSA and ~0.2-1.6 for OVA. They provide information about the relative rates of nitration or reaction probabilities for different tyrosine residues. In BSA, the tyrosine residue Y(161) was by far most reactive against NO(2)+O(3) and one of the four most reactive positions with regard to nitration by TNM. In OVA, all except one tyrosine residue detected in nitrated form exhibited similar reactivities. The observed nitration patterns show how the site selectivity of protein nitration depends on the nitrating agent, reaction conditions, and molecular structure of the protein (primary, secondary, and tertiary).