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

Abstract

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, Y162. The time-dependent increase in nitration at this site was measured based on the increasing abundance of the peptide 162YnLYEIAR168 and the corresponding decrease in the 162YLYEIAR168 peptide in in-gel trypsin digests. The peptide 66LVNEVTEFAK75, 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.

When is Mass Spectrometry Combined with Affinity Approaches Essential? A Case Study of Tyrosine Nitration in Proteins

“…For the ECP and EDN proteins, the identification of analogous specific Tyr-nitration sites is in agreement with the high degree of structural homology for both proteins [37]. These results demonstrate specific tyrosine nitrations in Eosinophil proteins and illustrate the proteolytic affinityextraction-MS as a highly efficient approach, far more specific and sensitive compared with LC-MS/MS approaches employed in previous studies [26,27] and in the identification of the self-nitration of eosinophil peroxidase (EPO) [38]. Moreover, the proteolytic affinity-extraction-MS of producing and submitting a peptide mixture is applicable to single proteins, protein complexes from biological material and protein bands from electrophoretic separations with comparable efficiency [28,36].…”

“…The affinity of the nitrated ECP peptide was found to be completely abolished upon replacing the positively charged residues Arg 28 , Arg 34 , Arg 36 , and Lys 38 by alanine, thus confirming the importance of cationic amino acids in the vicinity to the nitration site for affinity binding. Affinity determinations of the nitrated peptide in comparison to the intact ECP protein using the SAW biosensor provided dissociation constants of approximately 6 nM for the nitrated peptide ECP (24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41), and 28 nM for the intact ECP.…”

“…Thus, UV-MALDI-MS has been found to be critical for unequivocal identification of nitrations from biological samples due to the photochemical decomposition of nitrotyrosine residues [24,25]. In contrast, electrospray ionization mass spectrometry and IR-MALDI-MS have been shown to provide unambiguous identifications of NT-modified peptides [25][26][27]. In previous studies, different approaches of MS have been successfully employed for identifying tyrosine nitrations upon reaction with peroxynitrite in vitro [12,28] and in vivo [3].…”

Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS

“…Most MS methods for the quantitation of protein phosphorylation assume that MS is not quantitatively reproducible. However, there is increasing evidence that MS can provide some quantitation of protein abundances (22)(23)(24) and protein modifications (25,26) based on the ion signal intensities (ion current). These experiments show that appropriate normalization procedures are essential for deriving quantitative information without the use of stable isotopes.…”

Mass spectrometry in aging research

“…One strategy involves selective reduction of 3-nitrotyrosine to 3-aminotyrosine followed by covalent coupling to a cleavable, disulfide-containing, biotin affinity tag (Nikov et al, 2003). Quantitative analysis of 3-nitrotyrosine-containing peptides can be achieved by ESI-MS analysis using the native reference peptide (NRP) method, i.e., relative to the abundance of unmodified peptides of a given protein of interest (Willard et al, 2003). The ESI-MS n analysis of 3-nitrotyrosine-containing peptides yields unambiguous results, where the introduction of the nitro group increases the molecular weight of the original peptide by þ45 atomic mass units (amu).…”