A Method for Selective Enrichment and Analysis of Nitrotyrosine-Containing Peptides in Complex Proteome Samples

Abstract

Elevated levels of protein tyrosine nitration have been found in various neurodegenerative diseases and age-related pathologies. Until recently, however, the lack of an efficient enrichment method has prevented the analysis of this important low-level protein modification. We have developed a method that specifically enriches nitrotyrosine-containing peptides so that both nitrotyrosine peptides and specific nitration sites can be unambiguously identified with LC-MS/MS. The procedure consists of the derivatization of nitrotyrosine into free sulfhydryl groups followed by high efficiency enrichment of sulfhydryl-containing peptides with thiopropyl sepharose beads. The derivatization process includes: (1) acetylation with acetic anhydride to block all primary amines, (2) reduction of nitrotyrosine to aminotyrosine, (3) derivatization of aminotyrosine with N-Succinimidyl S-Acetylthioacetate (SATA), and (4) deprotection of S-acetyl on SATA to form free sulfhydryl groups. The high specificity of this method is demonstrated by the contrasting percentage of nitrotyrosine-derivatized peptides in the identified tandem mass spectra between enriched and unenriched samples. Global analysis of unenriched in vitro nitrated human histone H1.2, bovine serum albumin (BSA), and mouse brain homogenate samples had 9%, 9%, and 5.9% of identified nitrotyrosine-containing peptides, while the enriched samples had 91% , 62%, and 35%, respectively. Duplicate LC-MS/MS analyses of the enriched mouse brain homogenate identified 150 unique nitrated peptides covering 102 proteins with an estimated 3.3% false discovery rate.

Oxidation of proteins: Basic principles and perspectives for blood proteomics

“…Interestingly, Zhang et al [124] have recently introduced a methodology for selective isolation of nitrotyrosine-containing peptides: first, primary amines are acetylated, and free sulphydryls are blocked, nitrotyrosines are then reduced to aminotyrosine, which are then acetylthioacetylated, and the resulting group is deprotected to leave a free sulphydryl group, which in turn allows specific enrichment of peptides initially containing 3-nitrotyrosine. Doing so, authors were able to dramatically enrich peptides containing 3-nitrotyrosine compared to a direct mass spectrometric analysis.…”

Protein 3-Nitrotyrosine in Complex Biological Samples: Quantification by High-Pressure Liquid Chromatography/Electrochemical Detection and Emergence of Proteomic Approaches for Unbiased Identification of Modification Sites

“…This limitation was clearly appreciated by Zhang et al (2007), who modified the Nikov et al strategy and thereby enabled the identification of a number of protein nitration sites in a peroxynitrite-treated brain extract. The Zhang et al (2007) protocol involves protein digestion with trypsin as a first step, followed by reduction and alkylation of all free cysteine thiols with 5 mM dithiothreitol and 20 mM iodoacetamide.…”

“…The Zhang et al (2007) protocol involves protein digestion with trypsin as a first step, followed by reduction and alkylation of all free cysteine thiols with 5 mM dithiothreitol and 20 mM iodoacetamide. In this procedure, all free amines are then blocked by acetylation, using a 200-fold molar excess of acetic anhydride in ammonium bicarbonate buffer, pH 8.5.…”

“…While this is recognized as a significant advance over the Nikov et al (2003) strategy, which was only used to identify a single nitrated protein spiked into plasma, it fell short of the goal of identifying endogenously nitrated proteins. Furthermore, the 150 protein nitration sites identified by Zhang et al (2007) represented only 35% of the sequenced spectra-the other 65% of peptides that were "enriched" by the affinity protocol were nonnitrated. The relative inefficiency of the Zhang et al (2007) strategy for the enrichment of in vitro nitrated peptides is likely to preclude the effective use of this approach to capture and sequence peptides in protein digests containing low endogenous levels of tyrosine nitration, estimated at <0.1% of total tyrosine residues.…”

“…Furthermore, the 150 protein nitration sites identified by Zhang et al (2007) represented only 35% of the sequenced spectra-the other 65% of peptides that were "enriched" by the affinity protocol were nonnitrated. The relative inefficiency of the Zhang et al (2007) strategy for the enrichment of in vitro nitrated peptides is likely to preclude the effective use of this approach to capture and sequence peptides in protein digests containing low endogenous levels of tyrosine nitration, estimated at <0.1% of total tyrosine residues. We predict that the high false-positive peptide capture rate with the Zhang et al (2007) method lies in the choice of affinity tag.…”