Site-Specific Quantitation of Protein Nitration Using Liquid Chromatography/Tandem Mass Spectrometry

The native reference peptide (NRP) method has been adapted to the measure of the degree of protein nitration at a specific tyrosine residue. In these experiments, human serum albumin was modified in a myeloperoxidase-mediated reaction in the presence of nitrite, with nitration detected predominantly at one site, Y^162. The time-dependent increase in nitration at this site was measured based on the increasing abundance of the peptide 162Y^nLYEIAR^168 and the corresponding decrease in the 162YLYEIAR^168 peptide in in-gel trypsin digests. The peptide 66LVNEVTEFAK^75, also formed in the tryptic digest, was used as the native reference peptide. Quantitation was achieved by determining the chromatographic peak area of the two analyte peptides relative to the native reference peptide by LC/tandem mass spectrometric analyses with selected reaction monitoring. The NRP results were validated by correlation to the time-dependent increase in total protein-nitrotyrosine content determined by Western blot analysis. The precision and limit of detection of the assay were also evaluated and were found to be approximately 10% (relative standard deviation) and 5 fmol on-column, respectively. These results demonstrate the utility of the NRP method for quantitative analyses of posttranslation modifications, in terms of broad applicability, ease of experimental design, sensitivity, and precision.