微量元素铁与蛋白质酪氨酸硝化

蛋白质酪氨酸硝化是一氧化氮依赖的氧化应激的生物标志。蛋白质硝化将会直接影响蛋白质的催化活性、细胞信号传递和细胞骨架结构，导致相关病症的发生发展。本文介绍了铁在不同酪氨酸硝化途径中的作用，结果提示体内的微量铁对蛋白质硝化起着重要作用。     

蛋白质酪氨酸硝化的研究

蛋白质酪氨酸硝基化是一种重要的蛋白质翻译后修饰,与多种病症相关。经由过氧亚硝酸根(ONOO-)和NO2-/H2O2/血红素过氧化物酶体系是促使蛋白质硝化最主要的两种途径,其反应为自由基机理。本文对体内蛋白质硝基化的途径、机制及其生物学意义作了综述,指出蛋白质的硝化具有选择性,特定酪氨酸残基发生硝化能够改变蛋白质的结构和功能,影响其免疫应答和可能涉及的信号转导过程,从而具有重要的生物学意义。     

蛋白质酪氨酸硝化的研究

蛋白质酪氨酸硝基化是一种重要的蛋白质翻译后修饰,与多种病症相关.经由过氧亚硝酸根(ONOO-)和NO2-/H2O2/血红素过氧化物酶体系是促使蛋白质硝化最主要的两种途径,其反应为自由基机理.本文对体内蛋白质硝基化的途径、机制及其生物学意义作了综述,指出蛋白质的硝化具有选择性,特定酪氨酸残基发生硝化能够改变蛋白质的结构和功能,影响其免疫应答和可能涉及的信号转导过程,从而具有重要的生物学意义.     

蛋白质酪氨酸硝化的研究

蛋白质酪氨酸硝基化是一种重要的蛋白质翻译后修饰，与多种病症相关。经由过氧亚硝酸根（ONOO-）和NO2^-／H2O2／血红素过氧化物酶体系是促使蛋白质硝化最主要的两种途径，其反应为自由基机理。本文对体内蛋白质硝基化的途径、机制及其生物学意义作了综述，指出蛋白质的硝化具有选择性，特定酪氨酸残基发生硝化能够改变蛋白质的结构和功能，影响其免疫应答和可能涉及的信号转导过程，从而具有重要的生物学意义。     

糖尿病和硝化条件下胰岛素受体及底物酪氨酸磷酸化的研究

利用新颖的定量核磁共振(³¹P NMR)法和免疫印迹法研究了四氧嘧啶诱导的糖尿病状态下以及酪氨酸经过氧亚硝酸根供体SIN-1硝化条件下大鼠肝脏胰岛素受体(IR)的自磷酸化和受体底物1(IRS1)的磷酸化。结果表明,四氧嘧啶诱导的糖尿病大鼠肝脏中IR自磷酸化水平削弱了,硝化对大鼠肝脏中IR自磷酸化的影响依赖于SIN-1浓度,根据IRS1磷酸化位点基序设计的多肽的硝化完全抑制了其磷酸化,提示酪氨酸硝化可能干扰胰岛素磷酸化信号通路。     

谷胱甘肽和Ebselen对胰岛素硝化的抑制及其相互作用机理的研究

生物体内NO和超氧阴离子快速反应生成的过氧亚硝酸根离子(ONOO^-，peroxynitrite)是一种强细胞毒性物质，它诱导蛋白质酪氨酸残基硝化是其损伤生物系统的重要途径之一。为了探讨谷胱甘肽和ebselen对胰岛素硝化的抑制及其相互作用机理，采用UV-Vis、HPLC和ESI-MS等方法，研究了ONOO^-对胰岛素的硝化作用、小分子抗氧化剂谷胱甘肽(GSH)和ebselen对ONOO^-硝化胰岛素的影响以及它们之间的相互作用。结果表明单独的GSH和ebselen对ONOO^--引发的胰岛素硝化均有明显的抑制，而作为谷胱甘肽过氧化物酶(GPx)的底物GSH 与GPx的模型化合物ebselen之间存在相互拮抗作用，经过对其产物分析，确定其机理是GSH和ebselen能够直接反应生成一种加合物，从而抑制了GSH和ebselen各自的抗硝化能力。     

含苯并噁唑的L-酪氨酸衍生物的合成

从L-酪氨酸乙酯出发,经氨基保护、硝化、还原反应制得3-氨基-L-酪氨酸乙酯(4);4与苯甲醛或取代苯甲醛完成环化反应,合成了6个新的含苯并噁唑的L-酪氨酸衍生物,其结构经1H NMR,13C NMR,IR和元素分析表征.     

HPLC-ESI-MS/MS测定胰岛素硝基化修饰产物

糖尿病患者组织中存在过量表达的ONOO-及3-硝基酪氨酸(3-NT),ONOO-对胰岛素的硝化修饰可能是糖尿病发病的重要原因.本实验采用HPLC-ESI-MS/MS及分子模拟法,研究了ONOO-与胰岛素反应的硝化产物、B链酪氨酸位点的硝化次序及硝化产物结构特征.     

基于离心式富集装置的酪氨酸磷酸肽分析新方法

酪氨酸磷酸化及其相应激酶活性的研究在抗肿瘤药物靶点的研发中具有重要意义.由于酪氨酸磷酸化仅占蛋白质总磷酸化含量的不足0.1%,因此规模化的酪氨酸磷酸化鉴定面临着重大技术挑战.本研究构建了TiO2串联C18反相填料的离心式富集装置,结合抗体免疫沉淀法,建立了酪氨酸磷酸肽的富集策略.此新型富集装置由吸头、适配器和离心(EP)管组成,将TiO2富集磷酸肽和C18填料反相分离磷酸肽有机结合,以离心的方式进行样品的上样、清洗、洗脱和分离,再通过抗酪氨酸磷酸化抗体进一步特异性富集酪氨酸磷酸肽,从而实现了酪氨酸磷酸肽的高效富集和大规模质谱鉴定.通过离心式富集装置简化了实验步骤,减少了样品损失和人为因素干扰;而且离心式、平行化的样品处理方式可显著提高分析通量.将此策略成功用于小鼠肝脏蛋白质酪氨酸磷酸化肽段的富集和质谱鉴定,在5 mg鼠肝蛋白中共鉴定出967个酪氨酸磷酸化位点,对应545个蛋白质,显示了其在蛋白质组学研究中的应用潜力.     

酪氨酸磷酸化多肽的化学选择性富集

酪氨酸激酶在生物分子的信号转导中起着非常重要的作用,目前除抗体技术外尚无有效的化学方法能够实现对酪氨酸磷酸化蛋白或多肽的选择性富集,然而抗体通常成本较高,而且往往会有模体序列的选择性识别。本文发展了一种基于化学反应的酪氨酸磷酸化肽段的选择性富集,该方法利用了β消除反应只能发生在丝氨酸和苏氨酸磷酸化多肽的特性,以反相选择方法,从而实现对酪氨酸磷酸化肽段的选择性富集。以标准多肽对其反应效率和回收率进行了考察,20分钟内丝氨酸磷酸化多肽的β消除反应效率可达99%以上,而同时酪氨酸磷酸化肽段可保持70%的回收率。进一步以六种标准蛋白混合物的酶解产物对其进行考察,经β消除反应和亲和富集之后,只有酪氨酸磷酸化多肽可以被检测出来,该方法为蛋白质酪氨酸磷酸化的分析提供了一种新的手段。     

Analysis of nitrated proteins and tryptic peptides by HPLC-chip-MS/MS: site-specific quantification,nitration degree,and reactivity of tyrosine residues

The reaction products and pathways of protein nitration were studied with bovine serum albumin (BSA) and ovalbumin (OVA) nitrated by liquid tetranitromethane (TNM) or by gaseous nitrogen dioxide and ozone (NO₂ + O₃). Native and nitrated proteins were enzymatically digested with trypsin, and the tryptic peptides were analyzed by high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) using a chip cube nano-flow system (Agilent). Upon nitration by TNM, up to ten of 17 tyrosine residues in BSA and up to five of ten tyrosine residues in OVA could be detected in nitrated form. Upon nitration by NO₂ + O₃, only three nitrated tyrosine residues were found in BSA. The nitration degrees of individual nitrotyrosine residues (ND_Y) were determined by site-specific quantification and compared to the total protein nitration degrees (ND) determined by photometric detection of HPLC-DAD. The slopes of the observed linear correlations between ND_Y and ND varied in the range of ~0.02–2.4 for BSA and ~0.2–1.6 for OVA. They provide information about the relative rates of nitration or reaction probabilities for different tyrosine residues. In BSA, the tyrosine residue Y₁₆₁ was by far most reactive against NO₂ + O₃ and one of the four most reactive positions with regard to nitration by TNM. In OVA, all except one tyrosine residue detected in nitrated form exhibited similar reactivities. The observed nitration patterns show how the site selectivity of protein nitration depends on the nitrating agent, reaction conditions, and molecular structure of the protein (primary, secondary, and tertiary).     

Mass spectrometry analysis of in vitro nitration of a recombinant human IgG1 monoclonal antibody

Nitration of a recombinant human monoclonal antibody was carried out in vitro by incubating the antibody with the nitrating reagent tetranitromethane (TNM). The susceptible sites of nitration were identified using high-performance liquid chromatography/mass spectrometry (HPLC/MS). In general, tyrosine residues in the variable domains of the antibody are more susceptible to nitration, while tyrosine residues in the constant domains are relatively resistant to nitration. However, one tyrosine residue in the CH1 domain and one tyrosine residue in the CH2 domain are highly susceptible to nitration. Interestingly, the susceptible tyrosine residue in the CH2 domain is followed by the conserved asparagine residue that is glycosylated.     

Evidence for the Prerequisite Formation of Phenoxyl Radicals in Radical-Mediated Peptide Tyrosine Nitration In Vacuo

The elementary mechanism of radical-mediated peptide tyrosine nitration, which is a hallmark of post-translational modification of proteins under nitrative stress in vivo, has been elucidated in detail by using an integrated approach that combines the gas-phase synthesis of prototypical molecular tyrosine-containing peptide radical cations, ion–molecule reactions, and isotopic labeling experiments with DFT calculations. This reaction first involves the radical recombination of ^.NO₂ towards the prerequisite phenoxyl radical tautomer of a tyrosine residue, followed by proton rearrangements, finally yielding the stable and regioselective 3-nitrotyrosyl residue product. In contrast, nitration with the π-phenolic radical cation tautomer is inefficient. This first direct experimental evidence for the elementary steps of the radical-mediated tyrosine nitration mechanism in the gas phase provides a fundamental insight into the regioselectivity of biological tyrosine ortho-nitration.     

Copper-catalyzed tyrosine nitration

Tyrosine nitration, often observed during neurodegenerative disorders under nitrative stress, is usually considered to be induced chemically either by nitric oxide and oxygen forming nitrogen dioxide or by the decomposition of peroxynitrite. It can also be induced enzymatically by peroxidases or superoxide dismutases in the presence of both hydrogen peroxide and nitrite forming nitrogen dioxide and/or peroxynitrite. In this study, the role of cupric ions for catalyzing tyrosine nitration in the presence of hydrogen peroxide and nitrite, by a chemical mechanism rather similar to enzymatic pathways where nitrite is oxidized to form nitrogen dioxide, was investigated by development of a microreactor also capable of acting as an emitter for electrospray ionization mass spectrometry analysis. Indeed, cupric ions and peptide-cupric ion complexes are found to be excellent Fenton catalysts, even better than Fe(III) or heme, for the formation of (?)OH radicals and/or copper(II)-bound (?)OH radicals from hydrogen peroxide. These radicals are efficiently scavenged by nitrite anions to form (?)NO(2) and by tyrosine to form tyrosine radicals, leading to tyrosine nitration in polypeptides. We also show that cupric ions can catalyze tyrosine nitration from nitric oxide, oxygen, and hydrogen peroxide as the formation of tyrosine radicals is increased in the presence of diffusible and/or copper(II) bound hydroxyl radicals. This study shows that copper has a polyvalent role in the processes of tyrosine nitration.     

Selective scavenging property of the indole moiety for the nitrating species of peroxynitrite

The inhibitory effect on tyrosine nitration and oxidation of peroxynitrite was evaluated for more than 40 reagents including natural and synthetic compounds, and the inhibiting efficiency of each compound for nitration was compared with that for oxidation, to characterize its property as a peroxynitrite scavenger. In the presence of various concentrations of testing compounds, the nitrating and oxidizing activities were measured by monitoring the formation of 3-nitrotyrosine and dityrosine with an HPLC-UV-fluorescence detector. The IC(50) values for nitration and oxidation were determined, and the ratio of these two IC(50) values was calculated for each compound. Although the IC(50) values varied from compound to compound, it was revealed that the ratio of two IC(50) values (IC(50) for oxidation/IC(50) for nitration) was 1 in almost all the compounds tested, except five indole derivatives (L-tryptophan, melatonin, 5-methoxytryptamine, tryptamine, and tetrahydro-beta-carboline) and one synthetic selenium-containing compound ((2R,3R,4S)-2-amino-3,4-dihydroxy-5-phenylselenopentan-1-ol, ADPP). The indole derivatives showed a specific inhibitory effect on tyrosine nitration without affecting the oxidation. ADPP was confirmed to have a preferable inhibitory activity for tyrosine oxidation. It was suggested that compounds showing an IC(50) value ratio of 1 scavenged the common species for nitration and oxidation, while the indole derivatives and ADPP preferably scavenged the nitrating and oxidizing species, respectively. From a stopped flow study, it was also revealed that the nitrotyrosine formation was relatively slow, unlike an OH radical reaction. These results imply that the peroxynirite reaction at least partly proceeds through specific species for nitration.     

Identification of nitrated tyrosine residues of protein kinase G-Iα by mass spectrometry

The nitration of tyrosine to 3-nitrotyrosine is an oxidative modification of tyrosine by nitric oxide and is associated with many diseases, and targeting of protein kinase G (PKG)-I represents a potential therapeutic strategy for pulmonary hypertension and chronic pain. The direct assignment of tyrosine residues of PKG-I has remained to be made due to the low sensitivity of the current proteomic approach. In order to assign modified tyrosine residues of PKG-I, we nitrated purified PKG-Iα expressed in insect Sf9 cells by use of peroxynitrite in vitro and analyzed the trypsin-digested fragments by matrix-assisted laser desorption/ionization–time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Among the 21 tyrosine residues of PKG-Iα, 16 tyrosine residues were assigned in 13 fragments; and six tyrosine residues were nitrated, those at Y71, Y141, Y212, Y336, Y345, and Y567, in the peroxynitrite-treated sample. Single mutation of tyrosine residues at Y71, Y212, and Y336 to phenylalanine significantly reduced the nitration of PKG-Iα; and four mutations at Y71, Y141, Y212, and Y336 (Y4F mutant) reduced it additively. PKG-Iα activity was inhibited by peroxynitrite in a concentration-dependent manner from 30 μM to 1 mM, and this inhibition was attenuated in the Y4F mutant. These results demonstrated that PKG-Iα was nitrated at multiple tyrosine residues and that its activity was reduced by nitration of these residues.     

Identification of Nitrotyrosine Containing Peptides using Combined Fractional Diagonal Chromatography (COFRADIC) and Off-Line Nano-LC-MALDI

Protein nitration take place on tyrosine residues under oxidative stress conditions and may influence a number of processes including enzyme activity, protein-protein interactions and phospho-tyrosine signalling pathways. Nitrated proteins have been identified in a number of diseases, however, the study of these proteins has been compromised by the lack of good methods for identifying nitrated proteins, their nitration sites and the level of nitration. Here, we present a method for identification of nitrated peptides that allows the site specific assignment of nitration, is easy to use and reproducible, and opens up for the possibility to quantify the level of nitration of specific peptides as function of different oxidative conditions, namely combined fractional diagonal chromatography (COFRADIC) in combination with off-line nano-LC-MALDI. We identify six nitrated peptides from in vitro nitrated bovine serum albumin and propose that automated COFRADIC using nano-LC and off-line MALDI-MS might be a possibility for identification of tyrosine nitrated proteins and the nitration sites in complex samples.     

Investigation of tyrosine nitration in proteins by mass spectrometry.

In vivo nitration of tyrosine residues is a post-translational modification mediated by peroxynitrite that may be involved in a number of diseases. The aim of this study was to evaluate possibilities for site-specific detection of tyrosine nitration by mass spectrometry. Angiotensin II and bovine serum albumin (BSA) nitrated with tetranitromethane (TNM) were used as model compounds. Three strategies were investigated: (i) analysis of single peptides and protein digests by matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping, (ii) peptide mass mapping by electrospray ionization (ESI) mass spectrometry and (iii) screening for nitration by selective detection of the immonium ion of nitrotyrosine by precursor ion scanning with subsequent sequencing of the modified peptides. The MALDI time-of-flight mass spectrum of nitrated angiotensin II showed an unexpected prompt fragmentation involving the nitro group, in contrast to ESI-MS, where no fragmentation of nitrated angiotensin II was observed. The ESI mass spectra showed that mono- and dinitrated angiotensin II were obtained after treatment with TNM. ESI-MS/MS revealed that the mononitrated angiotensin II was nitrated on the side-chain of tyrosine. The dinitrated angiotensin II contained two nitro groups on the tyrosine residue. Nitration of BSA was confirmed by Western blotting with an antibody against nitrotyrosine and the sites for nitration were investigated by peptide mass mapping after in-gel digestion. Direct mass mapping by ESI revealed that two peptides were nitrated. Precursor ion scanning for the immonium ion for nitrotyrosine revealed two additional partially nitrated peptides. Based on the studies with the two model compounds, we suggest that the investigation of in vivo nitration of tyrosine and identification of nitrated peptides might be performed by precursor ion scanning for the specific immonium ion at m/z 181.06 combined with ESI-MS/MS for identification of the specific nitration sites.