5,064 research outputs found
Top-Down Mass Analysis of Protein Tyrosine Nitration: Comparison of Electron Capture Dissociation with “Slow-Heating” Tandem Mass Spectrometry Methods
Tyrosine nitration in proteins is an important post-translational modification (PTM) linked to various pathological conditions. When multiple potential sites of nitration exist, tandem mass spectrometry (MS/MS) methods provide unique tools to locate the nitro-tyrosine(s) precisely. Electron capture dissociation (ECD) is a powerful MS/MS method, different in its mechanisms to the “slow-heating” threshold fragmentation methods, such as collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD). Generally, ECD provides more homogeneous cleavage of the protein backbone and preserves labile PTMs. However recent studies in our laboratory demonstrated that ECD of doubly charged nitrated peptides is inhibited by the large electron affinity of the nitro group, while CID efficiency remains unaffected by nitration. Here, we have investigated the efficiency of ECD versus CID and IRMPD for top-down MS/MS analysis of multiply charged intact nitrated protein ions of myoglobin, lysozyme, and cytochrome c in a commercial Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. CID and IRMPD produced more cleavages in the vicinity of the sites of nitration than ECD. However the total number of ECD fragments was greater than those from CID or IRMPD, and many ECD fragments contained the site(s) of nitration. We conclude that ECD can be used in the top-down analysis of nitrated proteins, but precise localization of the sites of nitration may require either of the “slow-heating” methods
Bidimensional Tandem Mass Spectrometry for Selective Identification of Nitration Sites in Proteins.
Nitration of protein tyrosine residues is very often regarded as a molecular signal of peroxynitrite formation during development, oxidative stress, and aging. However, protein nitration might also have biological functions comparable to protein phosphorylation, mainly in redox signaling and in signal transduction. The major challenge in the proteomic analysis of nitroproteins is the need to discriminate modified proteins, usually occurring at substoichiometric levels from the large amount of nonmodified proteins. Moreover, precise localization of the nitration site is often required to fully describe the biological process. Existing methodologies essentially rely on immunochemical techniques either using 2D-PAGE fractionation in combination with western blot analyses or exploiting immunoaffinity procedures to selectively capture nitrated proteins. Here we report a totally new approach involving dansyl chloride labeling of the nitration sites that rely on the enormous potential of MSn analysis. The tryptic digest from the entire protein mixture is directly analyzed by MS on a linear ion trap mass spectrometer. Discrimination between nitro- and unmodified peptide is based on two selectivity criteria obtained by combining a precursor ion scan and an MS3 analysis. This new procedure was successfully applied to the identification of 3-nitrotyrosine residues in complex protein mixtures
Tyrosine Nitration of Voltage-dependent Anion Channels in Cardiac Ischemia-reperfusion: Reduction by Peroxynitrite Scavenging
Excess superoxide (O2−) and nitric oxide (NO) forms peroxynitrite (ONOO−) during cardiac ischemia reperfusion (IR) injury, which in turn induces protein tyrosine nitration (tyr-N). Mitochondria are both a source of and target for ONOO−. Our aim was to identify specific mitochondrial proteins that display enhanced tyr-N after cardiac IR injury, and to explore whether inhibiting O2−/ONOO− during IR decreases mitochondrial protein tyr-N and consequently improves cardiac function. We show here that IR increased tyr-N of 35 and 15 kDa mitochondrial proteins using Western blot analysis with 3-nitrotyrosine antibody. Immunoprecipitation (IP) followed by LC–MS/MS identified 13 protein candidates for tyr-N. IP and Western blot identified and confirmed that the 35 kDa tyr-N protein is the voltage-dependent anion channel (VDAC). Tyr-N of native cardiac VDAC with IR was verified on recombinant (r) VDAC with exogenous ONOO−. We also found that ONOO− directly enhanced rVDAC channel activity, and rVDAC tyr-N induced by ONOO− formed oligomers. Resveratrol (RES), a scavenger of O2−/ONOO−, reduced the tyr-N levels of both native and recombinant VDAC, while L-NAME, which inhibits NO generation, only reduced tyr-N levels of native VDAC. O2− and ONOO− levels were reduced in perfused hearts during IR by RES and L-NAME and this was accompanied by improved cardiac function. These results identify tyr-N of VDAC and show that reducing ONOO− during cardiac IR injury can attenuate tyr-N of VDAC and improve cardiac function
Striatal neuroinflammation promotes parkinsonism in rats
The specific role of neuroinflammation in the pathogenesis of Parkinson's disease remains to be fully elucidated. By infusing lipopolysaccharide (LPS) into the striatum, we investigated the effect of neuroinflammation on the dopamine nigrostriatal pathway. Here, we report that LPS-induced neuroinflammation in the striatum causes progressive degeneration of the dopamine nigrostriatal system, which is accompanied by motor impairments resembling parkinsonism. Our results indicate that neurodegeneration is associated with defects in the mitochondrial respiratory chain related to extensive S-nitrosylation/nitration of mitochondrial proteins. Mitochondrial injury was prevented by treatment of L-N^6^-(l-iminoethyl)-lysine, an inducible nitric oxide synthase (iNOS) inhibitor, suggesting that iNOS-derived NO is responsible for mitochondrial dysfunction. Furthermore, the nigral dopamine neurons exhibited intracytoplasmic [alpha]-synuclein and ubiquitin accumulation. These results demonstrate that degeneration of nigral dopamine neurons by neuroinflammation is associated with mitochondrial malfunction induced by NO-mediated S-nitrosylation/nitration of mitochondrial proteins
Increased glycation and oxidative damage to apolipoprotein B100 of LDL cholesterol in patients with type 2 diabetes and effect of metformin
OBJECTIVE The aim of this study was to investigate whether apolipoprotein B100 of LDL suffers increased damage by glycation, oxidation, and nitration in patients with type 2 diabetes, including patients receiving metformin therapy.
RESEARCH DESIGN AND METHODS For this study, 32 type 2 diabetic patients and 21 healthy control subjects were recruited; 13 diabetic patients were receiving metformin therapy (median dose: 1.50 g/day). LDL was isolated from venous plasma by ultracentrifugation, delipidated, digested, and analyzed for protein glycation, oxidation, and nitration adducts by stable isotopic dilution analysis tandem mass spectrometry.
RESULTS Advanced glycation end product (AGE) content of apolipoprotein B100 of LDL from type 2 diabetic patients was higher than from healthy subjects: arginine-derived AGE, 15.8 vs. 5.3 mol% (P < 0.001); and lysine-derived AGE, 2.5 vs. 1.5 mol% (P < 0.05). Oxidative damage, mainly methionine sulfoxide residues, was also increased: 2.5 vs. 1.1 molar equivalents (P < 0.001). 3-Nitrotyrosine content was decreased: 0.04 vs. 0.12 mol% (P < 0.05). In diabetic patients receiving metformin therapy, arginine-derived AGE and methionine sulfoxide were lower than in patients not receiving metformin: 19.3 vs. 8.9 mol% (P < 0.01) and 2.9 vs. 1.9 mol% (P < 0.05), respectively; 3-nitrotyrosine content was higher: 0.10 vs. 0.03 mol% (P < 0.05). Fructosyl-lysine residue content correlated positively with fasting plasma glucose. Arginine-derived AGE residue contents were intercorrelated and also correlated positively with methionine sulfoxide.
CONCLUSIONS Patients with type 2 diabetes had increased arginine-derived AGEs and oxidative damage in apolipoprotein B100 of LDL. This was lower in patients receiving metformin therapy, which may contribute to decreased oxidative damage, atherogenicity, and cardiovascular disease
Atmospheric chemistry of bioaerosols: heterogeneous and multiphase reactions with atmospheric oxidants and other trace gases.
Advances in analytical techniques and instrumentation have now established methods for detecting, quantifying, and identifying the chemical and microbial constituents of particulate matter in the atmosphere. For example, recent cryo-TEM studies of sea spray have identified whole bacteria and viruses ejected from ocean seawater into air. A focal point of this perspective is directed towards the reactivity of aerosol particles of biological origin with oxidants (OH, NO3, and O3) present in the atmosphere. Complementary information on the reactivity of aerosol particles is obtained from field investigations and laboratory studies. Laboratory studies of different types of biologically-derived particles offer important information related to their impacts on the local and global environment. These studies can also unravel a range of different chemistries and reactivity afforded by the complexity and diversity of the chemical make-up of these particles. Laboratory experiments as the ones reviewed herein can elucidate the chemistry of biological aerosols
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Nox2 dependent redox-regulation of microglial response to amyloid-β stimulation and microgliosis in aging
Microglia express constitutively a Nox2 enzyme that is involved in neuroinflammation by the
generation of reactive oxygen species (ROS). Amyloid β (Aβ) plays a crucial role in Alzheimer’s disease.
However, the mechanism of Aβ-induced microglial dysfunction and redox-regulation of microgliosis
in aging remains unclear. In this study, we examined Nox2-derived ROS in mediating microglial
response to Aβ peptide 1–42 (Aβ42) stimulation in vitro, in aging-associated microgliosis in vivo and in
post-mortem human samples. Compared to controls, Aβ42 markedly induced BV2 cell ROS production,
Nox2 expression, p47phox and ERK1/2 phosphorylation, cell proliferation and IL-1β secretion. All
these changes could be inhibited to the control levels in the presence of Nox2 inhibitor or superoxide
scavenger. Compared to young (3–4 months) controls, midbrain tissues from wild-type aging mice (20–
22 months) had significantly higher levels of Nox2-derived ROS production, Aβ deposition, microgliosis
and IL-1β production. However, these aging-related changes were reduced or absent in Nox2 knockout
aging mice. Clinical significance of aging-associated Nox2 activation, microgliosis and IL-1β production
was investigated using post-mortem midbrain tissues of humans at young (25–38 years) and old age
(61–85 years). In conclusion, Nox2-dependent redox-signalling is crucial in microglial response to Aβ42
stimulation and in aging-associated microgliosis and brain inflammation
Short-term alpha- or gamma-delta-enriched tocopherol oil supplementation differentially affects the expression of proinflammatory mediators: selective impacts on characteristics of protein tyrosine nitration in vivo
Development of an Analytical Assay for Electrochemical Detection and Quantification of Protein-Bound 3-Nitrotyrosine in Biological Samples and Comparison with Classical, Antibody-Based Methods.
Reactive oxygen and nitrogen species (RONS) cause oxidative damage, which is associated with endothelial dysfunction and cardiovascular disease, but may also contribute to redox signaling. Therefore, their precise detection is important for the evaluation of disease mechanisms. Here, we compared three different methods for the detection of 3-nitrotyrosine (3-NT), a marker of nitro-oxidative stress, in biological samples. Nitrated proteins were generated by incubation with peroxynitrite or 3-morpholino sydnonimine (Sin-1) and subjected to total hydrolysis using pronase, a mixture of different proteases. The 3-NT was then separated by high performance liquid chromatography (HPLC) and quantified by electrochemical detection (ECD, CoulArray) and compared to classical methods, namely enzyme-linked immunosorbent assay (ELISA) and dot blot analysis using specific 3-NT antibodies. Calibration curves for authentic 3-NT (detection limit 10 nM) and a concentration-response pattern for 3-NT obtained from digested nitrated bovine serum albumin (BSA) were highly linear over a wide 3-NT concentration range. Also, ex vivo nitration of protein from heart, isolated mitochondria, and serum/plasma could be quantified using the HPLC/ECD method and was confirmed by LC-MS/MS. Of note, nitro-oxidative damage of mitochondria results in increased superoxide (O2•-) formation rates (measured by dihydroethidium-based HPLC assay), pointing to a self-amplification mechanism of oxidative stress. Based on our ex vivo data, the CoulArray quantification method for 3-NT seems to have some advantages regarding sensitivity and selectivity. Establishing a reliable automated HPLC assay for the routine quantification of 3-NT in biological samples of cell culture, of animal and human origin seems to be more sophisticated than expected
Analysis of peroxynitrite-mediated post-translational modifications of caveolin-1
Caveolin-1 is an important protein in caveolae, which plays a role in cholesterol transport, signal transduction, and transcytosis and tumor suppression. Caveolin-1 is found in endothelial cells, smooth muscle cells and adipocytes. The main focus of this study is to investigate the peroxynitrite-mediated in vitro post-translational modifications (PTMs) of caveolin-1.
Bovine brain was used to isolate caveolin-1 as an initial step for isolation method development. Density gradient centrifugation was used to isolate caveolin-1 from bovine brain. From the isolate, caveolin-1α, caveolin-1β isomers and caveolin dimer were identified by western blotting with anti-caveolin monoclonal antibody. Glutathione S-transferase (GST)-caveolin fusion protein was used to isolate caveolin-1 and used for in vitro experiments in this study.
During normal and pathological conditions, endothelial cells are subjected to locally generated reactive oxygen species such as peroxynitrite. Peroxynitrite is capable of modifying amino acids such as tyrosine, cysteine, tryptophan and methionine.
Peroxynitrite mediated tyrosine nitration of caveolin-1 was detected by SDS-PAGE followed by western blotting with anti-nitrotyrosine monoclonal antibody. The approach used to identify potentially modified peptide sequences of caveolin-1 was ESI-MS/MS. Fluorometry was used to detect formation of dityrosine.
Caveolin-1 was treated with different concentrations of peroxynitrite in caveolin- under the physiological conditions and found that caveolin-1 form dimer and oligomer under the physiological conditions. The stability of caveolin-1 dimer and oligomer suggests that the coupling mechanism could most likely be occurred via a covalent bond. Western blotting with anti-nitrotyrosine monoclonal antibody revealed the formation of nitrotyrosine upon the exposure to peroxynitrite.
In this study, we report the nitration of specific tyrosine residues of caveolin-1 for the first time. ESI-MS/MS analysis revealed that peroxynitrite can selectively nitrate Tyr6 and Tyr14 located in the tryptic peptide YVDSEGHLYTVPIR under physiological conditions. Caveolin-1 can form dityrosine upon exposure to peroxynitrite as shown by fluorometry. Oxidative and nitrative modifications due to the reaction of peroxynitrite with caveolin-1 may lead to several pathological conditions. Our study can provide authentic standards of modified proteins, which will be used to determine post-translational modifications of caveolin-1 in vivo
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