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

Abstract

Post-translational protein nitration has attracted interest owing to its involvement in cellular signaling, effects on protein function and potential as biomarker of nitroxidative stress. We describe a procedure for enriching nitropeptides for mass spectrometry (MS)-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 (SPAER), and those peptides with amino groups will be selectively and covalently captured. Release of the peptides on hydrolysis with trifluoroacetic acid (TFA) 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 SPAER takes about 1 d. Enrichment of nitropeptides requires about 2 d, and sample preparations need 1–30 h, depending on the experimental design. LC-MS/MS assays take from 4 h to several days and data processing can be done in 1–7 d.

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Figure 1: Post-translational protein nitration.
Figure 2: Reaction scheme for the preparation of SPAER.
Figure 3: Schematic illustration of the SPAER-based enrichment procedure.
Figure 4: Scheme illustrating Steps 30–41 of the protocol.
Figure 5: Example anticipated results21.
Figure 6: Example of relative quantification by light and heavy dimethyl labeling via the SPAER-based nitropeptide enrichment33.

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Acknowledgements

This research was supported by the US National Institutes of Health (grant no. AG025384) and the Robert A. Welch Foundation (endowment no. BK-0031).

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K.P.-T. designed and synthesized SPAER, as well as optimized experimental conditions for applications. J.G. performed all experiments; J.G. and L.P. analyzed data; and L.P. supervised the project. All coauthors contributed to the writing of the manuscript.

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Correspondence to Laszlo Prokai.

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Prokai, L., Guo, J. & Prokai-Tatrai, K. Selective chemoprecipitation to enrich nitropeptides from complex proteomes for mass-spectrometric analysis. Nat Protoc 9, 882–895 (2014). https://doi.org/10.1038/nprot.2014.052

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