应用激光解吸质谱和十二烷基硫酸钠—聚丙烯酰胺凝胶电泳分析蛇毒？…

应用基质辅助激光解吸电离飞行时间质谱（ＭＡＬＤＩ－ＴＯＦ－ＭＳ）和ＳＤＳ－聚丙烯酰胺凝胶电流对吉林省两地市的同种白眉蝮蛇蛇毒中具有抗栓塞药效的精氨酸酯酶进行了分析了分析和比较。ＭＡＬＤＩ－ＴＯＦ－ＭＳ法具有快速、准确度高、灵敏度高的优点,两种方法结合,互为补充,取得了令人满意的结果。ＭＡＬＤＩ－ＴＯＦ－ＭＳ完全可以直接用作蛇毒成分分离过程中重要的研究手段。     

MALDI-TOF-MS在生物化学和高分子化学中的应用

基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)这一先进技术是80年代末发展起来的一个质谱学分支,它的诞生无疑为国内诸多领域注入了一丝新鲜的活力.自从带基质的激光解吸电离飞行时间质谱技术问世以来,具有良好的"软电离"性质对杂质的包容性以及可直接分析混合物而无需预先分离等特点,已广泛地应用于生物化学、高分子化学、有机化学、金属有机化学、药学等领域,显示出独特的潜力和应用前景.本文讨论了MALDI-TOF-MS技术在生物化学和高分子化学领域中的应用.     

白眉蝮蛇蛇毒和蛇毒酶的基质辅助激光解吸电离飞行时间质谱法研究

应用基质辅激光解吸电离飞行时间质谱（ＭＡＬＤＩ－ＴＯＦ－ＭＳ）法对长白山眉蝮蛇蛇毒和纯化得到的两种蛇毒酶进行了研究,得到了它们的分子质量并验证了纯度。同时还考察了不同产地的蛇毒、蛇毒蛋白浓度以及基质对分析结果的影响,实验结果表明ＭＡＬＤＩ－ＴＯＦ－ＭＳ法是检测蛋白纯化过程和分析蛋白相对分子质量十分有效的手段。     

基体辅助激光解吸电离质谱法对DNA样品的高分辨率测定

基体辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)法因其具有测定质量范围大、灵敏度高、速度快及精确度好等优点,近年来已成为测定多肽、蛋白质、核酸、多糖等生物大分子分子量及其一级结构的有力工具.     

聚芳醚酮环状低聚物的基质辅助激光解吸电离质谱表征

采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术分别对2种合成的聚芳醚酮环状低聚物进行了分析研究 ,并讨论了低聚物中不同聚合度离子组分的分布规律。实验结果表明MALDI-TOF-MS是分析环状低聚物直观、准确、快速的分析工具。     

白眉蝮蛇蛇毒及蛇毒酶的激光解吸飞行时间质谱研究

Perkins等[1]用MALDI/TOF/MS对不同种类蛇毒中神经毒素进行了分析,彭嘉柔等[2,3]对江浙蝮蛇和蛇毒粗组份进行了初步质谱表征.但蛇毒蛋白纯化困难,因此对单一组份的研究较少且不系统.本文以白眉蝮蛇蛇毒(AgkistrodonblomhoffiiUssurensis,ABUV)为原料,纯化得到了精氨酸酯酶(Arginineesterase,AEase)、磷脂酶A2(PhospholipaseA2,PLA2)、纤溶酶(fibrinolyticenzyme)和L-氨基酸氧化酶(L-aminoacidoxidase),并且用MALDI/TOF/MS法对它们和蛇毒粗毒进行了系统研究.1　实验部分1.1　仪器和药品　激光解吸质谱仪为美国Molecular公司L…     

蛇毒蛋白质中金属离子及其配位化学的研究

蛇毒蛋白质中金属离子及其配位化学的研究刘清亮吴双顶张祖德余华明（中国科学技术大学应用化学系安徽合肥２３００２６）在生命体内，金属离子能参与许多重要的生物化学反应，其实质就是金属离子与生物大分子的相互作用。蛇毒可以用作药物，具有抗凝、止血、抗癌和镇痛等...     

长白山白眉蝮蛇蛇毒酶的基质辅助激光解吸质谱分析

用基质辅助激光解吸飞行时间质谱法对长白山眉蝮蛇蛇毒所含４种主要酶：磷脂酶Ａ２,精氨酸酯酶,纤溶酶及Ｌ－氨基酸氧化酶进行了纯度鉴定和分子量测定,结果表明ＭＡＬＤＩ－ＴＯ－ＦＭＸ具有灵敏度高,分辨能力强,分析时间短及样品用量少等优点。用ＭＡＬＤＩ－ＴＯＦＭＳ法分析蛇毒酶的纯度和分子量简捷,快速且重现性好,是ＳＤＳ聚丙烯酰胺凝胶电泳所无法比拟的。     

介孔沸石材料负载传统基质用于激光解吸电离-飞行时间质谱分析小分子化合物

考察了介孔沸石材料负载传统有机基质α-氰基-4-羟基桂皮酸(CHCA)用于基质辅助激光解吸电离-飞行时间质谱(MALDI-TOF-MS)分析多肽Substance P和氟喹诺酮类药物等小分子的效果.在相同的MALDI-TOF-MS质谱条件下,与传统CHCA进行了比较,同时分别考察了不同硅铝比(SiO2/Al2 O3)的ZSM-5以及不同介孔大小的Beta与ZSM-5沸石载体对Substance P的检测效果.结果表明,沸石负载CHCA新型复合基质具有抑制碱金属离子峰、消除干扰碎片离子、简化与改善质谱图、提高离子化效率等优点.实验结果表明,沸石表面酸性越强,有力介孔能够充分包裹CHCA分子,则复合基质抑制干扰碎片和提高离子化效率的能力越高.复合基质成功应用于复杂样品中恩诺沙星与诺氟沙星药物小分子的MALDI-TOF-MS检测.     

基于特征肽的超高效液相色谱-串联质谱法检测矛头蝮蛇蛇毒种属来源及类凝血酶含量

蛇毒血凝酶类药物是以蝮蛇蛇毒为原料制备的止血药,主要活性成分为蛇毒类凝血酶(svTLEs)。不同蛇种来源的svTLEs结构不同,止血机制不同,药理作用也存在差异,因此准确鉴别蛇毒种属来源和svTLEs含量对于保障该类产品的质量至关重要。研究基于蛋白质组学技术,筛选出了具有种属特异性的矛头蝮蛇svTLE特征肽,并建立了基于特征肽的超高效液相色谱-串联质谱(UHPLC-MS/MS)检测矛头蝮蛇蛇毒种属来源及类凝血酶含量的方法。采用胰蛋白酶对纯化的矛头蝮蛇svTLE进行酶解,利用纳升液相色谱-四极杆/静电场轨道阱高分辨质谱(Nano LC-Q-Exactive-MS)和Proteome Discoverer 2.2软件分别进行多肽的检测和鉴定,通过BLAST搜索与Uniprot数据库对比分析,筛选出具有种属特异性的矛头蝮蛇svTLEs特征肽“EAYNGLPAK”。针对该特征肽对酶解温度、酶解时间和酶用量等样品前处理方法进行了优化,利用超高效液相色谱-串联质谱,以m/z 481.9>315.2和481.9>485.2作为检测离子对,采用ESI⁺模式进行了多反应...     

Antihaemorrhagic potentials of Fagonia cretica against Naja naja karachiensis (black Pakistan cobra) venom

Plants have been extensively used as a remedy for the treatment of snake bites. The aim of this study was to determine the antivenom potentials of methanolic extract from the aerial parts (leaves and twigs) of Fagonia cretica L. on a haemorrhage induced by venom from Naja naja karachiensis. The haemorrhagic response of venom was dose dependent from 0.1 to 4.0?μg per 1.5?μL phosphate buffer saline (PBS) on vitelline veins of fertilised hens' eggs in their shells. The extract effectively eliminated and neutralised, in a dose-dependent manner, the haemorrhagic activity of snake venom. The minimum effective neutralising dose of F. cretica extract was found to be 15?μg per 1.5?μL PBS. The extract possesses potentials as haemorrhagic inhibitor against snake venom compared to the standard antiserum and various plants reported in the literature. This study also provides a scientific base for the use of F. cretica in traditional medicine for the treatment of snake bite.     

Cytotoxicity of Silica Nanoparticles with Transcaucasian Nose-Horned Viper, <Emphasis Type="Italic">Vipera ammodytes transcaucasiana</Emphasis>, Venom on U87MG and SHSY5Y Neuronal Cancer Cells

Highly bioactive compounds of the snake venom make them particular sources for anticancer agent development. They contain very rich peptide-protein structures. Therefore, they are very susceptible to environmental conditions such as temperature, pH, and light. In this study, Vipera ammodytes transcaucasiana venom was encapsulated in PAMAM-G4 dendrimer by sol-gel method in order to prevent degradation of venom contents from the environmental conditions. For this purpose, nanoparticles were prepared by sol-gel methodology and SEM analyses were performed. U87MG and SHSY5Y neuronal cancer cell lines were treated with different concentrations of venom-containing nanoparticles and cytotoxicity was determined by MTT assay. IC₅₀ values of nanoparticles with snake venom were calculated as 37.24 and 44.64 μg/ml for U87MG and SHSY5Y cells, respectively. The IC₅₀ values of nanoparticles with snake venom were calculated as 10.07 and 7.9 μg/ml for U87MG and SHSY5Y cells, respectively. As a result, nanoparticles with V. a. transcaucasiana venom showed remarkably high cytotoxicity. Encapsulation efficiency of nanoparticles with 1 mg/ml snake venom was determined as %67 via BCA? protein analysis. In conclusion, this method is found to be convenient and useful for encapsulating snake venom as well as being suitable for drug delivery systems.     

Identification evidence unraveled by strict proteomics rules toward forensic samples

Snake venom is a complex mixture of proteins and peptides secreted by venomous snakes from their poison glands. Although proteomics for snake venom composition, interspecific differences, and developmental evolution has been developed for a decade, current diagnosis or identification techniques of snake venom in clinical intoxication and forensic science applications are mainly dependent on morphological and immunoassay. It could be expected that the proteomics techniques directly offer great help. This work applied a bottom-up proteomics method to identify proteins’ types and species attribution in suspected snake venom samples using ultrahigh-performance liquid chromatography–quadrupole-electrostatic field Orbitrap tandem mass spectrometric technique, and cytotoxicity assay was amended to provide a direct evidence of toxicity. Toward the suspicious samples seized in the security control, sample pretreatment (in-sol and in-gel digestion) and data acquisition (nontargeted and targeted screening) modes complemented and validated each other. We have implemented two consequent approaches in identifying the species source of proteins in the samples via the points of venom proteomics and strict forensic identification. First, we completed a workflow consisting of a proteomics database match toward an entire SWISS-PROT (date 2018-11-22) database and a result-directed specific taxonomy database. The latter was a helpful hint to compare master protein kinds and reveal the insufficiency of specific venom proteomics characterization rules. Second, we suggested strict rules for protein identification to meet the requirements of forensic science on improved identification correctness, that is, (1) peptide spectrum matches confidence, peptide confidence, and protein confidence were both high (with the false-discovery ratio less than 1%); (2) the number of unique peptides was more than or equal to two in one protein, and (3) within unique peptides, which at least 75% of the ∆m/z of the matched y and b ions were less than 5 ppm. We identified these samples as cobra venom containing 10 highly abundant proteins (P00597, P82463, P60770, Q9YGI4, P62375, P49123, P80245, P60302, P01442, and P60304) from two snake venom protein families (acid phospholipase A2 and three-finger toxins), and the most abundant proteins were cytotoxins.     

疑似蛇毒液样品的纳升级超高效液相色谱-高分辨质谱分析鉴定

针对5个疑似蛇毒毒液及其沾染样品,基于纳升级超高效液相色谱-四极杆-静电场轨道阱高分辨质谱（Nano LC-MS/HRMS）技术,结合尺寸排阻色谱分离,建立了一种蛋白质种类及物种归属的严格鉴定方法。5个样品经尺寸排阻色谱分离后均得到3个洗脱峰,分别冻干后以胰蛋白酶进行溶液内酶解处理并进行液相色谱-高分辨质谱分析鉴定。首先采用全扫描-数据依赖型MS/MS（Full MS/dd MS²）采集模式对样品中的肽段信息进行非靶向采集,依次与Swiss-Prot、蛇亚目（Serpentes）、游蛇科（Colubroidea）、眼镜蛇科（Elapidae）、眼镜蛇亚科（Elapinae）、眼镜蛇属（Naja）蛋白质数据库逐级收缩比对;再筛选符合肽谱匹配度、肽段错误发现率小于1%和特征肽段数目大于等于2的蛋白质,共鉴定到32种蛋白质均来自中华眼镜蛇（Naja atra）,可归属于Naja atra的10个家族,主要为三指毒素、金属蛋白酶、磷脂酶A2等。最后,采用平行反应监测模式选取每种蛋白质的两条特征肽段进行靶向验证,当两条特征肽段均满足“至少75%的y⁺和b⁺离子的Δm/z小于5 ppm”时,方认为鉴定到了样品中的某一蛋白质。最终鉴定出5个样品均含有Naja atra蛇毒。此鉴定方法研究系统、严格,可为蛇毒中毒司法鉴定以及毒药物研究等提供有效的技术支持。     

A strategy for the enrichment and characterization of disulfide bond-contained proteins from Chinese cobra (Naja atra) venom

A new strategy combined gold-coated magnetic nanocomposites assisted enrichment with mass spectrometry was developed for the characterization of disulfide bond-contained proteins from Chinese cobra (Naja atra) venom. In this work, core-shell nanocomposites were synthesized by the seed-mediated growth method and used for the enrichment of snake venom proteins containing disulfide bonds. A total of 3545 tryptic digested peptides derived from 96 venom proteins in Naja atra venom were identified. The venom proteins comprised 14 toxin families including three-finger toxins, phospholipase A₂, snake venom metalloproteinase, cobra venom factor, and so forth. Extra 16 venom proteins were detected exclusively in the nanocomposites set, among which 11 venom proteins were from the three-finger toxins family. In the present study, the proposed simple and efficient protocol replaced the tedious and laborious technologies commonly used for pre-separating crude snake venom, suggesting widely implementation in low-abundance or trace disulfide bond-contained proteins or peptides characterization.     

Dacin,one metalloproteinase from <Emphasis Type="Italic">Deinagkistrodon acutus</Emphasis> venom inhibiting contraction of mouse ileum muscle

Background

Mice were bitten by five-pace vipers (Deinagkistrodon acutus), and then envenomed. It was well-known that the snake venom mainly disturbed the blood homeostasis of the envenomed victims. Ocassionally, we found that the venom of D. acutus could inhibit the contraction tension of mouse ileum, so in this study we aimed to identify the active component inhibiting the contraction tension of mouse ileum in the snake venom.

Results

The active component inhibiting the contraction tension of mouse ileum, designated as Dacin, was isolated from D. acutus venom, purified to protein homogeneity and composed of a single peptide chain, about 23 kDa analyzed by SDS-PAGE, and 22, 947. 9 Da measured by MALDI-TOF-MS. Not only the results of its PMF blasted by Mascot indicated that Dacin may be one snake venom metalloproteinase (SVMP), but also the results of the biochemical and in-vivo assays as follow demonstrated that it was one SVMP: it cleaved Aα and Bβ chains, not Cγ of bovine fibrinogen within 1 h, and also hydrolyzed fibrin polymer; besides its fibrino(geno)lytic activities were strongly inhibited by β- mercaptoethanol, EDTA and EGTA; and it could induce a hemorrhagic reaction under the dorsal skin of mouse. In the isolated tissue assays, Dacin caused the concentration-dependent and time-dependent inhibitory actions on the spontaneous contraction tension of the ileum smooth muscle of mouse, and the inhibitory effects were irreversible.

Conclusions

Taken together, for the first time one active component (Dacin, a SVMP) that irreversibly inhibited the spontaneous contraction tension of mouse ileum has been isolated and identified from D. acutus venom. The findings may provide not only a new insight for toxicological researches on SVMPs and venoms of the vipers, but also a reference for clinicians to treat the snake-bitten victims. However, Dacin’s inhibitory molecular mechanism will be further studied in the future.

     

Analysis of the Composition of Deinagkistrodon acutus Snake Venom Based on Proteomics,and Its Antithrombotic Activity and Toxicity Studies

There is a strong correlation between the composition of Deinagkistrodon acutus venom proteins and their potential pharmacological effects. The proteomic analysis revealed 103 proteins identified through label-free proteomics from 30 different snake venom families. Phospholipase A2 (30.0%), snaclec (21.0%), antithrombin (17.8%), thrombin (8.1%) and metalloproteinases (4.2%) were the most abundant proteins. The main toxicity of Deinagkistrodon acutus venom is hematotoxicity and neurotoxicity, and it acts on the lung. Deinagkistrodon acutus venom may have anticoagulant and antithrombotic effects. In summary, the protein profile and related toxicity and pharmacological activity of Deinagkistrodon acutus venom from southwest China were put forward for the first time. In addition, we revealed the relationship between the main toxicity, pharmacological effects, and the protein components of snake venom.     

金属离子对蛇毒蛋白生物活性及结构效应的影响

从旅顺产白眉蝮蛇(Gloydius blomhoffii brevicaudus, GBB) 蛇毒中纯化得到了3种电泳和质谱纯蛋白活性组分, 其酶解肽段采用高效液相色谱-电喷雾串联质谱(HPLC-nESI-MS/MS)进行序列测定, 与其它同源性蛇毒蛋白氨基酸序列比对发现, 3种蛋白为新的蛇毒磷脂酶A₂、类凝血酶和金属蛋白酶, 分别将其命名为GBB-bPLA₂, GBB-TLE和GBB-MP. 电感耦合等离子体发射光谱法(ICP-AES) 测得每个GBB-bPLA₂和GBB-MP中含有一个Ca²⁺; 每个GBB-TLE中含有2个Zn²⁺. Ca²⁺可分别使GBB-bPLA₂和GBB-MP的荧光发射波长向短波方向移动2.0和1.6 nm, 使二者的荧光发射强度提高14.0%和11.0%; Ca²⁺的存在可显著提高二者的热稳定性, 使GBB-bPLA₂和GBB-MP的热变性温度分别提高1.5和2.0 ℃. Zn²⁺使GBB-TLE的荧光发射强度增高4.3%, 但对GBB-TLE的酯酶水解活性、荧光发射波长和热变性温度无显著影响. 金属离子的存在能够不同程度地影响蛇毒蛋白结构热稳定性, 但对蛇毒蛋白生物活性的作用则不同.