Selective chemoprecipitation to enrich nitropeptides from complex proteomes for mass-spectrometric analysis

Abstract

Posttranslational protein nitration has attracted interest due to its involvement in cellular signaling, effects on protein function, and as a potential biomarker of nitroxidative stress. We describe a procedure for enriching nitropeptides for mass spectrometry-based proteomics that is a simple and reliable alternative to immunoaffinity-based methods. The starting material for this procedure is a proteolytic digest. The peptides are reacted with formaldehyde and sodium cyanoborohydride to dimethylate all the N-terminal and side-chain amino groups. Sodium dithionite is added subsequently to reduce the nitro groups to amines; in theory, the only amino groups present will have originally been nitro groups. The peptide sample is then applied to a solid-phase active ester reagent, and those peptides with amino groups will be selectively and covalently captured. Release of the peptides on hydrolysis with trifluoroacetic acid results in peptides that have a 4-formyl-benzamido group where the nitro group used to be. In qualitative setups, the procedure can be used to identify proteins modified by reactive nitrogen species and to determine the specific sites of their nitration. Quantitative measurements can be performed by stable-isotope labeling of the peptides in the reductive dimethylation step. Preparation of the solid-phase active ester reagent takes about 1 day. Enrichment of nitropeptides requires about 2 days, and sample preparations need 1 to 30 h varying based on experimental design. LC–MS/MS assays take from 4 h to several days and data processing can be done 1 to 7 days.

Mass spectrometry‐based retina proteomics

“…After demonstrating a possible role of nitroxidative protein nitration in normal retinal physiology through an immunoblotting‐based 2D‐GE study (Miyagi et al, 2002), anti‐nitrotyrosine immunoprecipitation from diabetic rat retina and RMG cells were used to enable their analyses by shotgun LC‐ESI‐MS/MS with focus on identifying nitroproteins (Zhan et al, 2008). However, all reported nitropeptides were misidentified, highlighting the importance of using efficient enrichment methods with rigorous validations for the purpose (Prokai et al, 2014; Prokai‐Tatrai et al, 2011; Stevens, Prokai‐Tatrai, et al, 2008).…”

Uncommon posttranslational modifications in proteomics: ADP‐ribosylation, tyrosine nitration, and tyrosine sulfation

“…Using acetylation to block the primary amines decreases the ionization efficiency of modified peptides by removing the protonation sites and can be mitigated by using methylation (Guo et al, 2012; Prokai et al, 2014; Prokai‐Tatrai et al, 2011). Additionally, reduction of 3‐nTyr to 3‐AT using dithionite leads to the formation of a more favorable and acidic side‐product (sulfated 3‐aminotyrosine) that cannot be further derivatized with enrichment tags, resulting in fewer identifications of nitropeptides (Ghesquière et al, 2009).…”

Direct detection of nitrotyrosine-containing proteins using an aniline-based oxidative coupling strategy

“…8 These include Western blotting with anti-Y-NO 2 antibodies, 9 direct analysis using mass spectrometry, 10 and chemical detection methods based on biotinylation, 11 resin capture, 12,13 and fluorophore formation. 14–16 .…”