蛋白质酪氨酸硝化检测方法

蛋白质酪氨酸硝化是一种重要的蛋白质翻译后的选择性修饰,其产物3－硝基酪氨酸可以作为检测细胞和组织损伤的一个生物标志。本文详细介绍了分析检测蛋白质酪氨酸硝化的各种方法,主要包括免疫法、分光光度法、色谱法、质谱法、电泳法等,并对其检测方法的发展趋势进行了评述和展望。     

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.     

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.     

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。     

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。     

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.     

铁卟啉与活性氮的相互作用

在活性氮(RNS)与蛋白质的相互作用过程中,含铁卟啉蛋白/铁卟啉可以催化RNS转化为惰性的NO3-,阻止蛋白质的硝化;同时,含铁卟啉蛋白/铁卟啉也可通过高价铁氧化物及.NO2的产生来加速蛋白质的硝化。生理条件下,血红蛋白、肌红蛋白等含铁卟啉蛋白可清除多余的RNS;氧化应激条件下,过氧化物酶等含铁卟啉蛋白可以不同的方式催化加速RNS对蛋白质的硝化。     

HPLC法对过亚硝酸根抑制剂筛选的研究

过亚硝酸根是由体内一氧化氮和超氧根阴离子反应生成的一种细胞毒性物质,能和许多生物大分子反应,如DNA、蛋白质和脂质等.本文通过高效液相色谱方法来研究槲皮素和黄酮对ONOO-诱导的DNA碱基修饰以及酪氨酸硝化的抑制作用,结果表明槲皮素抑制效果显著,而黄酮几乎没有效果.本实验的意义还在于可以将本方法推广到其他过亚硝酸根抑制剂的筛选中,成为一种比较有效且灵敏的抑制剂评价方法.     

硝酸铁对水杨酸甲酯的硝化及位置选择性

用硝酸铁对水杨酸甲酯硝化合成了3-硝基水杨酸甲酯和5-硝基水杨酸甲酯。 考察了反应时间、反应物配比、反应溶液酸碱性对产物收率和位置选择性的影响。 得出较好的反应条件为：反应时间3 h,n(硝酸铁)∶n(水杨酸甲酯)=2∶3。 少量硝酸有利于硝基化反应,且优先生成5-硝基水杨酸甲酯,而碱性不利于硝基化反应,且优先生成3-硝基水杨酸甲酯。 用量化计算解释了硝化反应的位置选择性机理。     

6-氯-2(1 H)-喹喔酮合成方法的改进

设计了以对氯苯胺和氯乙酰氯为原料,经缩合、硝化、还原、环合、氧化五步制得6-氯-2(1H)-喹喔酮的位置选择性合成方法,并对以氯乙酰氯作为苯胺的氨基保护试剂进行硝化的反应和对含活泼氯的硝基化合物进行还原的反应进行了研究.研究结果表明以氯乙酰氯作为苯胺的氨基保护试剂进行硝化的反应符合一般的硝化定位规则,而含活泼氯的硝基化合物的还原在优化条件下以铁粉为还原剂可以高选择性获得目标产物.     

β-Nitro derivatives of iron corrolates

Two different methods for the regioselective nitration of different meso-triarylcorroles leading to the corresponding β-substituted nitrocorrole iron complexes have been developed. A two-step procedure affords three Fe(III) nitrosyl products-the unsubstituted corrole, the 3-nitrocorrole, and the 3,17-dinitrocorrole. In contrast, a one-pot synthetic approach drives the reaction almost exclusively to formation of the iron nitrosyl 3,17-dinitrocorrole. Electron-releasing substituents on the meso-aryl groups of the triarylcorroles induce higher yields and longer reaction times than what is observed for the synthesis of similar triarylcorroles with electron-withdrawing functionalities, and these results can be confidently attributed to the facile formation and stabilization of an intermediate iron corrole π-cation radical. Electron-withdrawing substituents on the meso-aryl groups of triarylcorrole also seem to labilize the axial nitrosyl group which, in the case of the pentafluorophenylcorrole derivative, results in the direct formation of a disubstituted iron μ-oxo dimer complex. The influence of meso-aryl substituents on the progress and products of the nitration reaction was investigated. In addition, to elucidate the most important factors which influence the redox reactivity of these different iron nitrosyl complexes, selected compounds were examined by cyclic voltammetry and thin-layer UV-visible or FTIR spectroelectrochemistry in CH(2)Cl(2).     

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).     

Nonacid nitration of benzenedicarboxylic and naphthalenecarboxylic acid esters

When treated with nitrogen dioxide in the presence of ozone and a catalytic amount of iron(III) chloride in inert organic solvent at -10 to +5 degrees C, benzenedicarboxylic acid diesters 1, 4, and 6 underwent smooth nitration to give the corresponding mononitro derivatives 2/3, 5, and 7, respectively, in good yield (kyodai nitration). Naphthalenecarboxylic acid esters 8 and 11 and naphthalene-1,8-dicarboxylic acid diester 16 were similarly nitrated in the absence of catalyst to give the expected nitro compounds 9/10, 12-15, and 17-22, respectively. Different from conventional nitration based on the combined use of concentrated nitric and sulfuric acids, no hydrolytic cleavage of the ester function was observed under these conditions. The isomer distribution has been determined for the nitration of naphthalenecarboxylic acid esters 8, 11, and 16, and spectral data were collected for less common nitro derivatives. A unique changeover of the orientation mode observed in the kyodai nitration of diester 16, from the initial exclusive meta to the final meta/para, has been discussed in terms of the competition between the electrophilic substitution process involving the nitronium ion (NO2+) and the addition-elimination sequence involving the nitrogen trioxide radical (*NO3).     

Quantification of nitrotyrosine in nitrated proteins

For kinetic studies of protein nitration reactions, we have developed a method for the quantification of nitrotyrosine residues in protein molecules by liquid chromatography coupled to a diode array detector of ultraviolet-visible absorption. Nitrated bovine serum albumin (BSA) and nitrated ovalbumin (OVA) were synthesized and used as standards for the determination of the protein nitration degree (ND), which is defined as the average number of nitrotyrosine residues divided by the total number of tyrosine residues in a protein molecule. The obtained calibration curves of the ratio of chromatographic peak areas of absorbance at 357 and at 280 nm vs. nitration degree are nearly the same for BSA and OVA (relative deviations <5%). They are near-linear at low ND (< 0.1) and can be described by a second-order polynomial fit up to $ {\hbox{ND}} = 0.5\left( {{R^2} > 0.99} \right) $ {\hbox{ND}} = 0.5\left( {{R^2} > 0.99} \right) . A change of chromatographic column led to changes in absolute peak areas but not in the peak area ratios and related calibration functions, which confirms the robustness of the analytical method. First results of laboratory experiments confirm that the method is applicable for the investigation of the reaction kinetics of protein nitration. The main advantage over alternative methods is that nitration degrees can be efficiently determined without hydrolysis or digestion of the investigated protein molecules.     

When is Mass Spectrometry Combined with Affinity Approaches Essential? A Case Study of Tyrosine Nitration in Proteins

Tyrosine nitration in proteins occurs under physiologic conditions and is increased at disease conditions associated with oxidative stress, such as inflammation and Alzheimer??s disease. Identification and quantification of tyrosine-nitrations are crucial for understanding nitration mechanism(s) and their functional consequences. Mass spectrometry (MS) is best suited to identify nitration sites, but is hampered by low stabilities and modification levels and possible structural changes induced by nitration. In this insight, we discuss methods for identifying and quantifying nitration sites by proteolytic affinity extraction using nitrotyrosine (NT)-specific antibodies, in combination with electrospray-MS. The efficiency of this approach is illustrated by identification of specific nitration sites in two proteins in eosinophil granules from several biological samples, eosinophil-cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). Affinity extraction combined with Edman sequencing enabled the quantification of nitration levels, which were found to be 8?% and 15?% for ECP and EDN, respectively. Structure modeling utilizing available crystal structures and affinity studies using synthetic NT-peptides suggest a tyrosine nitration sequence motif comprising positively charged residues in the vicinity of the NT- residue, located at specific surface- accessible sites of the protein structure. Affinities of Tyr-nitrated peptides from ECP and EDN to NT-antibodies, determined by online bioaffinity- MS, provided nanomolar K_D values. In contrast, false-positive identifications of nitrations were obtained in proteins from cystic fibrosis patients upon using NT-specific antibodies, and were shown to be hydroxy-tyrosine modifications. These results demonstrate affinity- mass spectrometry approaches to be essential for unequivocal identification of biological tyrosine nitrations.     

Mass spectrometric characterization of photooxidative protein modifications in Arabidopsis thaliana thylakoid membranes

Oxidative and nitrosative stress leaves footprints in the plant chloroplast in the form of oxidatively modified proteins. Using a mass spectrometric approach, we identified 126 tyrosine and 12 tryptophan nitration sites in 164 nitrated proteolytic peptides, mainly from photosystem I (PSI), photosystem II (PSII), cytochrome b(6) /f and ATP-synthase complexes and 140 oxidation products of tyrosine, tryptophan, proline, phenylalanine and histidine residues. While a high number of nitration sites were found in proteins from four photosynthetic complexes indicating that the nitration belongs to one of the prominent posttranslational protein modifications in photosynthetic apparatus, amino acid oxidation products were determined mostly in PSII and to a lower extent in PSI. Exposure of plants to light stress resulted in an increased level of tyrosine and tryptophan nitration and tryptophan oxidation in proteins of PSII reaction center and the oxygen-evolving complex, as compared to low light conditions. In contrast, the level of nitration and oxidation of these amino acid residues strongly decreased for all light-harvesting proteins of PSII under the same conditions. Based on these data, we propose that oxidative modifications of proteins by reactive oxygen and nitrogen species might represent an important regulatory mechanism of protein turnover under light stress conditions, especially for PSII and its antenna proteins.