Dataset Information

Discovery of nitrotyrosine-containing proteins and peptides by antibody-based enrichment strategies

ABSTRACT: Nitrotyrosine is a modification in proteins caused by oxidative reactions with reactive nitrogen species. It's commonly associated with oxidative stress and linked to various health conditions like inflammation, neurodegeneration, cardiovascular disease, and cancer. However, detecting nitrotyrosine-modified proteins is challenging due to their low levels in biological samples. To make this detection more comprehensive, we optimized an immunoprecipitation-based method for both proteins and peptides using a cell line model. We tested the efficiency of four different commercially available monoclonal antibodies for enriching nitrotyrosine-modified proteins and peptides. Through LC-MS/MS analysis, we identified 1,377 nitrotyrosine-containing peptides from protein-based enrichment and 1,624 from peptide-based enrichment. However, we noticed a bias in the peptides enriched; a significant portion (37-65%) had nitrotyrosine at the beginning of the peptide when using the peptide-based approach, whereas this bias was minimal (<9%) with the protein-based immunoprecipitation. In total, we found 2,605 nitrotyrosine-containing peptides, with over 2,000 of them not previously reported. To validate our findings, we synthesized 110 of these peptides and analyzed them with LC-MS/MS. Additionally, we confirmed the presence of nitrotyrosine modifications in PKM and EF2 proteins in peroxynitrite-treated samples through immunoblot analysis. This extensive dataset and workflow will aid further exploration of nitrotyrosine as an oxidative modification in various contexts.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): B Cell

SUBMITTER: Akhilesh Pandey

LAB HEAD: Akhilesh Pandey

PROVIDER: PXD046168 | Pride | 2024-08-29

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	NT-Protein_IP_Abcam_R1.raw	Raw
	NT-Protein_IP_Abcam_R2.raw	Raw
	NT-Protein_IP_Abcam_R3.raw	Raw
	NT-Protein_IP_Invitrogen_R1.raw	Raw
	NT-Protein_IP_Invitrogen_R2.raw	Raw

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Publications

Exploration of Nitrotyrosine-Containing Proteins and Peptides by Antibody-Based Enrichment Strategies.

Bhat Firdous A FA Mangalaparthi Kiran K KK Ding Husheng H Jain Anu A Hsu Joel-Sean JS Peterson Jane A JA Zenka Roman M RM Mun Dong-Gi DG Kandasamy Richard K RK Pandey Akhilesh A

Molecular & cellular proteomics : MCP 20240210 3

Nitrotyrosine, or 3-nitrotyrosine, is an oxidative post-translational modification induced by reactive nitrogen species. Although nitrotyrosine is considered a marker of oxidative stress and has been associated with inflammation, neurodegeneration, cardiovascular disease, and cancer, identification of nitrotyrosine-modified proteins remains challenging owing to its low stoichiometric levels in biological samples. To facilitate a comprehensive analysis of proteins and peptides containing nitrotyr ...[more]

PMID: 38342410

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