Isolation of nitrotyrosine-containing peptides by using an insoluble-antibody column.
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ABSTRACT:
SUBMITTER: Helman M
PROVIDER: S-EPMC1178258 | biostudies-other | 1971 Dec
REPOSITORIES: biostudies-other
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Project description: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 nitrotyrosine, we optimized an immunoprecipitation-based enrichment workflow using a cell line model. The identification of proteins and peptides containing nitrotyrosine residues was carried out after peroxynitrite treatment of cell lysates, which generated modified nitrotyrosine residues on susceptible sites on proteins. We evaluated the efficacy of enriching nitrotyrosine-modified proteins and peptides by employing four different commercially available monoclonal antibodies directed against nitrotyrosine. LC-MS/MS analysis resulted in the identification of 1377 and 1624 nitrotyrosine-containing peptides from protein- and peptide-based enrichment experiments, respectively. Although the yield of nitrotyrosine-containing peptides was higher in experiments where peptides rather than proteins were enriched, we found a substantial proportion (37-65%) of identified nitrotyrosine-containing peptides contained nitrotyrosine at the N-terminus. However, in protein-based immunoprecipitation <9% of nitrotyrosine-containing peptides had nitrotyrosine modification at the N-terminus of the peptide. Overall, our study resulted in the identification of 2603 nitrotyrosine-containing peptides of which >2000 have not previously been reported. We synthesized 101 novel nitrotyrosine-containing peptides identified in our analysis and analyzed them by LC-MS/MS to validate our findings. We have confirmed the validity of 70% of these peptides, as they demonstrated a similarity score exceeding 0.7 when compared to peptides identified through experimental methods. Finally, we also validated the presence of nitrotyrosine modification on PKM and EF2 proteins in peroxynitrite-treated samples by immunoblot analysis. The large catalog presented in this study along with the workflow should facilitate the investigation of nitrotyrosine as an oxidative modification in a variety of settings in greater detail.
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Project description: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.
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Project description: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.
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Project description:A critical step in developing therapeutics for oxidative stress-related pathologies is the ability to determine which specific modified protein species are innocuous by-products of pathology and which are causative agents. To achieve this goal, technologies are needed that can identify, characterize and quantify oxidative post translational modifications (oxPTMs). Nanobodies (Nbs) represent exquisite tools for intracellular tracking of molecules due to their small size, stability and engineerability. Here, we demonstrate that it is possible to develop a selective Nb against an oxPTM protein, with the key advance being the use of genetic code expansion (GCE) to provide an efficient source of the large quantities of high-quality, homogenous and site-specific oxPTM-containing protein needed for the Nb selection process. In this proof-of-concept study, we produce a Nb selective for a 3-nitrotyrosine (nitroTyr) modified form of the 14-3-3 signaling protein with a lesser recognition of nitroTyr in other protein contexts. This advance opens the door to the GCE-facilitated development of other anti-PTM Nbs.
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