Proteomics analysis of protein tyrosine nitration, part 2
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)
TISSUE(S): Lung
SUBMITTER: 
Yingxin Zhao
LAB HEAD: Yingxin Zhao, Ph.D.
PROVIDER: PXD006427 | Pride | 2017-07-24
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| G1-NO-1.raw | Raw | |||
| G1-NO-2.raw | Raw | |||
| G1-NO-3-02.msf | Msf | |||
| G1-NO-3.raw | Raw | |||
| Glu.msf | Msf |
Items per page: 5 1 - 5 of 25 |
Publications
Selective Affinity Enrichment of Nitrotyrosine-Containing Peptides for Quantitative Analysis in Complex Samples.
Zhao Yingxin Y Zhang Yueqing Y Sun Hong H Maroto Rosario R Brasier Allan R AR
Journal of proteome research 20170717 8
Protein tyrosine nitration by oxidative and nitrate stress is important in the pathogenesis of many inflammatory or aging-related diseases. Mass spectrometry analysis of protein nitrotyrosine is very challenging because the non-nitrated peptides suppress the signals of the low-abundance nitrotyrosine (NT) peptides. No validated methods for enrichment of NT-peptides are currently available. Here we report an immunoaffinity enrichment of NT-peptides for proteomics analysis. The effectiveness of th ...[more]
Similar Datasets
Project description:Proteomics analysis of protein tyrosine nitration by LC-MS/MS
2017-07-24 | PXD005320 | Pride
Project description:Adhesion of T. cruzi trypomastigotes to components of the extracellular matrix (ECM) is an important step in mammalian host cell invasion. We have recently described a significant increase in the tyrosine nitration levels of histones H2A and H4 when trypomastigotes are incubated with components of the ECM. In this work, we used chromatin immunoprecipitation (ChIP) with an anti-nitro-tyrosine antibody followed by mass spectrometry to identify nitrated DNA binding proteins in T. cruzi and to detect alterations in nitration levels induced upon parasite incubation with the ECM. Histone H1, H2B, H2A and H3 were detected among the 9 most abundant nitrated DNA binding proteins using this proteomic approach. One nitrated tyrosine residue (Y29) was identified in Histone H2B in the MS/MS spectrum. In addition, we observed a significant increase in the nitration levels of histones H1, H2B, H2A and H4 upon parasite incubation with ECM. Finally, we used ChIP-Seq to map global changes in the DNA binding profile of nitrated proteins. We observed a significant change in the binding pattern of nitrated proteins to DNA after parasite incubation with ECM. This work provides the first global profile of nitrated DNA binding proteins in T. cruzi and additional evidence for modification in the nitration profile of histones upon parasite incubation with ECM. Our data also indicate that the interaction of the parasite with the ECM induces alterations in chromatin structure, possibly affecting nuclear functions.
2020-06-17 | GSE140245 | GEO
Project description:Adhesion of T. cruzi trypomastigotes to components of the extracellular matrix (ECM) is an important step in mammalian host cell invasion. We have recently described a significant increase in the tyrosine nitration levels of histones H2A and H4 when trypomastigotes are incubated with components of the ECM. In this work, we used chromatin immunoprecipitation (ChIP) with an anti-nitro-tyrosine antibody followed by mass spectrometry to identify nitrated DNA binding proteins in T. cruzi and to detect alterations in nitration levels induced upon parasite incubation with the ECM. Histone H1, H2B, H2A and H3 were detected among the 9 most abundant nitrated DNA binding proteins using this proteomic approach. One nitrated tyrosine residue (Y29) was identified in Histone H2B in the MS/MS spectrum. In addition, we observed a significant increase in the nitration levels of histones H1, H2B, H2A and H4 upon parasite incubation with ECM. Finally, we used ChIP-Seq to map global changes in the DNA binding profile of nitrated proteins. We observed a significant change in the binding pattern of nitrated proteins to DNA after parasite incubation with ECM. This work provides the first global profile of nitrated DNA binding proteins in T. cruzi and additional evidence for modification in the nitration profile of histones upon parasite incubation with ECM. Our data also indicate that the interaction of the parasite with the ECM induces alterations in chromatin structure, possibly affecting nuclear functions.
2021-09-09 | PXD016313 | Pride
Project description:Tyrosine nitration is a protein post-translational modification that is predominantly non-enzymatic and is observed to be increased under conditions of nitrosative stress and in numerous disease states. A small protein motif (14-18 amino acids) responsive to tyrosine nitration has been developed. In this design, nitrotyrosine replaced the conserved Glu12 of an EF-hand metal-binding motif. Thus, the non-nitrated peptide bound terbium weakly. In contrast, tyrosine nitration resulted in a 45-fold increase in terbium affinity. Nuclear magnetic resonance spectroscopy indicated direct binding of nitrotyrosine to the metal and EF-hand-like metal contacts in this designed peptide. Nitrotyrosine is an efficient quencher of fluorescence. To develop a sensor of tyrosine nitration, the initial design was modified to incorporate Glu residues at EF-hand positions 9 and 16 as additional metal-binding residues, to increase the terbium affinity of the peptide with unmodified tyrosine. This peptide with a tyrosine at residue 12 bound terbium and effectively sensitized terbium luminescence. Tyrosine nitration resulted in a 180-fold increase in terbium affinity ( Kd = 1.6 μM) and quenching of terbium luminescence. This sequence was incorporated as an encoded protein tag and applied as a turn-off fluorescent protein sensor of tyrosine nitration. The sensor was responsive to nitration by peroxynitrite, with fluorescence quenched upon nitration. The greater terbium affinity upon tyrosine nitration resulted in a large dynamic range and sensitivity to substoichiometric nitration. An improved approach for the synthesis of peptides containing nitrotyrosine was also developed, via the in situ silyl protection of nitrotyrosine. This work represents the first designed, encodable protein motif that is responsive to tyrosine nitration.
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Project description:Tyrosine Kinase Inhibitor Cardiotoxicity
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Project description:Nitrosative stress has been implicated in the pathogenesis of age related macular degeneration (AMD). Tyrosine nitration is a unique type of post translational modification that occurs in the setting of inflammation and nitrosative stress. To date, the significance and functional implications of tyrosine nitration of complement factor H (CFH), a key complement regulator in the eye has not been explored, and is examined in this study in the context of AMD pathogenesis. Sections of eyes from deceased individuals with AMD (n = 5) demonstrated the presence of immunoreactive nitrotyrosine CFH. We purified nitrated CFH from retinae from 2 AMD patients. Mass spectrometry of CFH isolated from AMD eyes revealed residues in domains critical for binding to heparan sulphate glycosaminoglycans (GAGs), lipid peroxidation by-products and complement (C) 3b were nitrated. Functional studies revealed that nitrated CFH did not bind to lipid peroxidation products, nor to the GAG of perlecan nor to C3b. There was loss of cofactor activity for Factor I mediated cleavage of C3b with nitrated CFH compared to non-nitrated CFH. CFH inhibits, but nitrated CFH significantly potentiates, the secretion of the pro-inflammatory and angiogenic cytokine IL-8 from monocytes that have been stimulated with lipid peroxidation by-products. AMD patients (n = 30) and controls (n = 30) were used to measure plasma nitrated CFH using a novel ELISA. AMD patients had significantly elevated nitrated CFH levels compared to controls (p = 0.0117). These findings strongly suggest that nitrated CFH contributes to AMD progression, and is a target for therapeutic intervention.
2017-08-29 | PXD005613 | Pride
Project description:The mechanisms of tyrosine nitration by peroxynitrous acid or nitrosoperoxycarbonate were investigated with the CBS-QB3 method. Either the protonation of peroxynitrite or a reaction with carbon dioxide gives a reactive peroxide intermediate. Peroxynitrous acid-mediated nitration of phenol occurs via unimolecular decomposition to give nitrogen dioxide and hydroxyl radicals. Nitrosoperoxycarbonate also undergoes unimolecular decomposition to give carbonate and nitrogen dioxide radicals. The reactions of tyrosine with the hydroxyl or carbonate radicals give a phenoxy radical intermediate. The reaction of the nitrogen dioxide with this radical intermediate followed by tautomerization gives nitrated tyrosine in both cases. According to CBS-QB3 calculations, the rate-limiting step for the nitration of phenol is the decomposition of peroxynitrous acid or nitrosoperoxycarbonate.
| S-EPMC2712443 | biostudies-literature
Project description:Using a three-pronged MS-based proteomic approach, we assessed the mode-of-action of four clinically-relevant tyrosine kinase inhibitors in an epithelial cancer cell line. Here we defined the: (I) semi-quantitative tyrosine kinome; (II) drug interactome; and (III) drug-induced effects on the tyrosine signalome. We first examined the entire proteome and kinome profile expressed in the A431 cell line, and determined the relative abundance of all possible protein targets of the tyrosine kinase inhibitors. We therefore utilized a ‘shot-gun’ approach that involved a multidimensional separation-based proteomic strategy. To ensure maximal protein identification, we reduced sample complexity by strong cation exchange (SCX) chromatography, and analyzed all fractions by LC-MS/MS. To determine the direct targets of the four selected TKIs, we subjected the proteins extracted from the A431 cancer cell line to drug-coupled affinity matrices. By comparing the pull-down results from a lysate with a second identical lysate to which 20 uM of the inhibitor had been added to block binding to the beads, we were able to identify selective binding interactors using LC-MS/MS. This differential affinity pull-downs approach allowed us to also quantitatively evaluate the binding affinity of the tyrosine kinases to each of the drugs. To evaluate the global tyrosine phosphorylation dynamics that occurs in A431 cells upon treatment with each of the four inhibitors, phosphotyrosine peptides were enriched and identified by quantitative mass spectrometry. For this purpose, we treated the cells with each inhibitor for 2 h at appropriate concentrations adopted from reported studies. Cells were lysed, and the proteins digested with the proteases Lys-C and trypsin. To allow quantitation of the phosphorylated peptides originating from the three different peptide pools (control/imatinib/dasatinib or control/bosutinib/nilotinib), we used stable isotope dimethyl labeling. Subsequently, tyrosine-phosphorylated peptides were enriched by immunoprecipitation and analyzed by LC-MS/MS.
2014-05-12 | PXD000681 | Pride
Project description:Tissues are exposed to exogenous and endogenous nitrogen dioxide ((·)NO(2)), which is the terminal agent in protein tyrosine nitration. Besides iron chelation, the hydroxamic acid (HA) desferrioxamine (DFO) shows multiple functionalities including nitration inhibition. To investigate mechanisms whereby DFO affects 3-nitrotyrosine (3-NT) formation, we utilized gas-phase (·)NO(2) exposures, to limit introduction of other reactive species, and a lung surface model wherein red cell membranes (RCM) were immobilized under a defined aqueous film. When RCM were exposed to ()NO(2) covered by +/- DFO: (i) DFO inhibited 3-NT formation more effectively than other HA and non-HA chelators; (ii) 3-NT inhibition occurred at very low[DFO] for prolonged times; and (iii) 3-NT formation was iron independent but inhibition required DFO present. DFO poorly reacted with (·)NO(2) compared to ascorbate, assessed via (·)NO(2) reactive absorption and aqueous-phase oxidation rates, yet limited 3-NT formation at far lower concentrations. DFO also inhibited nitration under aqueous bulk-phase conditions, and inhibited 3-NT generated by active myeloperoxidase "bound" to RCM. Per the above and kinetic analyses suggesting preferential DFO versus (·)NO(2) reaction within membranes, we conclude that DFO inhibits 3-NT formation predominantly by facile repair of the tyrosyl radical intermediate, which prevents (·)NO(2) addition, and thus nitration, and potentially influences biochemical functionalities.
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Project description:Diabetic retinopathy, a retinal vascular disease, is inhibited in animals treated with aminoguanidine, an inhibitor of inducible nitric-oxide synthase. This treatment also reduces retinal protein nitration, which is greater in diabetic rat retina than nondiabetic retina. As an approach to understanding the molecular mechanisms of diabetic retinopathy, we sought the identity of nitrotyrosine-containing proteins in retina from streptozotocin-induced diabetic rats and in a rat retinal Müller cell line grown in high glucose (25 mM). Anti-nitrotyrosine immunoprecipitation products from rat retina and Müller cells were analyzed by LC-MS/MS. Ten nitrated proteins in diabetic rat retina and three nitrated proteins in Müller cells grown in high glucose were identified; three additional nitrotyrosine-containing proteins were tentatively identified from diabetic retina. The identified nitrotyrosine-containing proteins participate in a variety of processes including glucose metabolism, signal transduction, and transcription/translation. Among the nitrated proteins were insulin-responsive glucose transporter type 4 (GLUT-4), which has been implicated previously in the pathogenesis of diabetes mellitus; exocyst complex component Exo70, which functions in insulin-stimulated glucose uptake of GLUT-4-containing vesicles; and fibroblast growth factor receptor 2, which influences retinal vascularization via fibroblast growth factor signaling. Nitration of tyrosine phosphorylation sites were identified in five proteins, including GLUT-4, exocyst complex component Exo70, protein-tyrosine phosphatase eta, sensory neuron synuclein, and inositol trisphosphate receptor 3. Quantitation of nitration and phosphorylation at common tyrosine modification sites in GLUT-4 and protein-tyrosine phosphatase eta from diabetic and nondiabetic animals suggests that nitration reduced tyrosine phosphorylation approximately 2X in these proteins from diabetic retina. The present results provide new insights regarding tyrosine nitration and its potential role in the molecular mechanisms of diabetic retinopathy.
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