武夷菌素高产基因工程菌株Streptomyces albulus OoWysR活性成分的分离鉴定

【背景】武夷菌素高产基因工程菌株Streptomyce albulus OoWysR具有明显的抑菌效果。【目的】明确S.albulus OoWysR的活性成分。【方法】采用柱层析法,利用大孔吸附树脂、离子交换树脂和高效液相色谱等对S.albulus OoWysR的活性成分进行分离纯化,经高分辨率电喷雾电离质谱(high-resolution electrospray ionization mass spectrometry,HR-ESI-MS)和核磁共振(nuclear magnetic resonance,NMR)等波谱技术对化合物化学结构进行鉴定,并通过生长速率法测定化合物的生物活性。【结果】从S.albulus OoWysR中分离鉴定出4个化合物,分别为对羟基苯甲酸(1)、吡咯-2-羧酸(2)、对羟基苯乙醇(3)和云南霉素(4)。化合物1对玉米弯孢病菌、番茄叶霉病菌、玉米小斑病菌和烟草赤星病菌具有一定的抑制作用;化合物2对番茄灰霉病菌、大豆菌核病菌、苹果腐烂病菌、玉米小斑病菌、小麦赤霉病菌、稻瘟病菌和烟草赤星病菌具有一定的抑制作用;化合物3对番茄灰霉病菌、苹果轮纹病菌、玉米小斑病...     

SELDI蛋白质芯片技术及其应用

随着人类基因组测序计划的完成,鉴定细胞内蛋白质表达、结构、功能及相互作用方式等成为后基因组时代的主要目标之一。为此,需要高通量的蛋白质组学研究的技术和方法。近年来出现的表面增强激光解吸／电离(surface—enhanced laser desorption／ionization,SELDI)蛋白质芯片技术是一种操作简单,方便快捷,样本需要量少,敏感性高,特异性强的高通量的研究蛋白组学的方法,在蛋白质功能分析、肿瘤标志物筛选、药物研发等方面具有广泛的应用前景。     

生物质谱技术在糖蛋白结构分析中的应用

生物质谱包括基质辅助激光解吸附飞行时间质谱及电喷雾质谱被广泛应用于生物样品如多肽、蛋白质及核酸的分析,由于这种具有软电离方式的质谱具有极高的灵敏度及准确度,目前也被成功地用于糖蛋白的结构分析,与普通的化学方法相比,质谱法快速、简单,结合网上数据库检索、凝集素亲和提取、二维凝胶电泳以及靶上直接酶切等新方法,可以提供糖蛋白的一级结构乃至高级结构的信息。     

生物质谱结合IMAC亲和提取和磷酸酶水解分析蛋白质磷酸化修饰

蛋白质磷酸化是生物体内非常重要的翻译后修饰方式 ,对磷酸化蛋白质的分析及磷酸化位点的确定有助于理解与其相关的生物功能。基质辅助激光解吸 /电离飞行时间质谱和电喷雾 四极杆 飞行时间质谱这两种生物质谱仪在蛋白质鉴定和翻译后修饰分析中发挥着重要作用。固相金属亲和色谱可选择性亲和提取肽混合物中的磷酸肽 ,结合磷酸酶水解实验和基质辅助激光解吸 /电离飞行时间质谱分析可确定肽混合物中的磷酸肽 ,最后用电喷雾 四极杆 飞行时间串联质谱分析磷酸肽的序列 ,结合数据库检索确定磷酸化位点。     

整体蛋白质电喷雾质谱原位解析

蛋白质组学系统研究了生物体蛋白质组,尤其是一定生理、病理条件下差异表达的蛋白;对蛋白质序列、翻译后修饰及其位置的定性鉴定可以帮助我们系统地了解蛋白质的结构和功能。随着软电离技术(如电喷雾电离技术)及高质量测量精度、高质量分辨质谱仪(如轨道阱质谱仪)的发展与相对普及,完整蛋白质的质谱表征(即所谓的自上而下蛋白质组学)已成为可能且渐渐流行起来;相应的数据库搜索引擎和蛋白质鉴定生物信息学工具也有了一定的进展。本文对作者研发的蛋白质电喷雾质谱原位解析算法"同位素质荷比及轮廓指纹比对"及整体蛋白质数据库搜索引擎"Protein Goggle2.0"(http://proteingoggle.tongji.edu.cn/)做一个概述。     

应用蛋白质组学技术筛选胃癌耐药相关蛋白质

胃癌多药耐药性是临床胃癌化疗失败最主要的原因之一,但其分子机制仍然不太清楚.为了寻找新的胃癌耐药相关的蛋白质,揭示胃癌多药耐药的分子机制,以胃癌细胞SGC7901和长春新碱诱导的耐药胃癌细胞SGC7901/VCR为研究对象,应用二维凝胶电泳(two-dimensionalelectrophoresis,2-DE)技术分离两种细胞的总蛋白质,图像分析识别差异表达的蛋白质点,基质辅助激光解吸电离飞行时间质谱(matrix-assistedlaserdesorption/ionizationtimeofflightmassspectrometry,MALDI-TOF-MS)及电喷雾电离串联质谱(electrosprayionizationtandemmassspectrometry,ESI-Q-TOF)对差异表达的蛋白质点进行鉴定,蛋白质印迹和实时RT-PCR验证部分差异蛋白质在两株细胞中的表达水平,反义核酸转染技术分析HSP27(heatshockprotein27,HSP27)高表达与SGC7901/VCR耐药的相关性.得到了分辨率较高、重复性较好的两株细胞系的二维凝胶电泳图谱,质谱分析共鉴定了24个差异蛋白质点,蛋白质印迹和实时RT-PCR验证了部分差异蛋白的表达水平,反义寡核苷酸抑制HSP27表达能增加SGC7901/VCR对长春新碱的敏感性.研究结果不仅提示这些差异蛋白质如HSP27,Sorcin等可能与胃癌的多药耐药相关,而且为揭示胃癌细胞的多药耐药性产生机制提供了线索.     

微生物学研究中的软电离质谱技术

包括基质辅助激光解吸电离（ＭＡＬＤＩ）和电喷雾（ＥＳＩ）在内的软电离质谱是最近发展起来的质谱技术,由于这些电离方式对样品的破坏性小,质量测定范围大,分子量测定准确,样品纯度要求不高很适合分析成分复杂的微生物样品,ＭＡＬＩＤＩ－ＴＯＦ－ＭＳ结合高分辨率的二维ＳＤＳ－ＰＡＧＥ可以分析１０＾－１２摩尔水平的蛋白,是细菌蛋白质研究过程中必不可少的工具。最近的研究工作表明,通过ＭＡＩＤＩ－ＴＯＦ－ＭＳ或ＨＰ     

MALDI-TOF-MS方法快速鉴定食品中空肠弯曲菌的技术研究

运用基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time of-flight mass spectrometry,MALDI-TOF-MS)技术快速鉴定食品中空肠弯曲菌.通过对该方法的样品前处理的选择、稳定性、特异性等方面进行研究,确定了方法...     

基于电喷雾电离串联质谱技术的植物脂质组学研究进展

研究表明,脂质不但参与植物的信号转导、小泡运输、细胞骨架重组等多种细胞过程,而且在植物的生长发育和胁迫反应中具有重要作用．但是脂质本身的多样性、复杂性、以及分析手段的滞后限制了人们对脂质的深入认识．电喷雾电离串联质谱(ESI-MS/MS)技术作为一种直接进样的高通量分析技术,能够在短时间内对大多数脂质的不同分子种进行定量分析,极大地方便了人们了解植物因环境变化和生长发育引起的组织内脂质分子种的微量变化．近年来,该技术在植物上的成功应用,推动植物脂质组学研究取得了重要进展,揭示出脂质在植物的逆境胁迫反应、防御反应中的多种功能,促进了植物脂质代谢相关基因的鉴定．而且,该技术与其他脂质分析技术结合,促使人们在脂质的分布、运输、转化和新脂质种类的鉴定方面有新的进展．概要介绍了ESI-MS/MS技术的特点,重点综述了该技术在植物脂质组学研究中的应用进展,并展望了该技术今后的发展方向.     

超高分辨率毛细管等电聚焦—电喷雾质谱联用方法观察蛋白质亚型

在线的毛细管等电聚焦 电喷雾质谱联用 ,作为一种二维的分离系统 ,对毛细管等电聚焦过程中形成的蛋白质亚型进行了分析。这种分析系统通过使用中性的涂层毛细管 (80cm长 )、动态的毛细管位置调整方法和鞘流液接口得以建立。蛋白质首先在毛细管等电聚焦过程中根据它们等电点的差异得到分离 ,然后被电喷雾质谱鉴定。已聚焦好的蛋白质区带通过结合阴极移动和重力移动的方法从毛细管中流出而进入质谱仪。由于在此特定情况下这种方法具有极高的分辨率 ,有三种血红蛋白A和镰刀型血红蛋白的亚型 (具有几乎相同的电荷分布和分子质量 ,但它们的等电点差异在 0 .0 4到 0 .0 8之间 )和两种乳球蛋白A的亚型 (等电点差异为 0 .6 )被检测到。这些蛋白质亚型的等电点、相对含量和分子质量都通过毛细管等电聚焦 电喷雾质谱联用方法同时得到了确定。     

Electrospray ionization mass spectrometric peptide mapping: a rapid, sensitive technique for protein structure analysis

Electrospray ionization mass spectrometric peptide mapping is demonstrated to be a useful new technique for protein structure analysis. The procedure involves the digestion of the protein with trypsin and subsequent analysis of the total unfractionated digest by electrospray ionization mass spectrometry. The utility of the technique for investigating protein structure is illustrated by a peptide mapping analysis of human apolipoprotein AI (Mr = 28 kDa). The technique is rapid, sensitive, and requires no prior separation of the peptides. The discrimination effects observed in other mass spectrometric methods are less important in the present procedure.     

Targeted metabolomic analysis of Escherichia coli by desorption electrospray ionization and extractive electrospray ionization mass spectrometry

Desorption electrospray ionization (DESI) was utilized to monitor the presence of targeted central carbon metabolites within bacterial cell extracts and the quench supernatant of Escherichia coli. The targeted metabolites were identified through tandem mass spectrometry (MS/MS) product ion scans using collision-induced dissociation in the negative ion mode. Picogram detection limits were achieved for a majority of the metabolites during MS/MS analysis of standard metabolite solutions. In a [U-(13)C]glucose pulse experiment, where uniformly labeled glucose was fed to E. coli, the corresponding fragment ions from labeled metabolites in extracts were generally observed. There was evidence of matrix effects including moderate suppression by other metabolites within the spectra of the labeled and unlabeled extracts. To improve the specificity and sensitivity of detection, optimized in situ ambient chemical reactions using DESI and extractive electrospray ionization (EESI) were carried out for targeted compounds. This study provides the first indication of the potential to perform in situ targeted metabolomics of a bacterial sample via ambient ionization mass spectrometry.     

Rapid detection of atrazine and its metabolite in raw urine by extractive electrospray ionization mass spectrometry

Herbicides such as atrazine are widely used in the biosphere. Urine analysis is usually performed to evaluate the toxicological effects associated with atrazine exposure. A simple procedure based on the extractive electrospray ionization mass spectrometry (EESI-MS) method was established to detect atrazine and its metabolites in undiluted raw urine without sample pretreatment. A 4.3 × 10⁻¹⁴ g atrazine in spiked raw urine was detected and identified by EESI/MS/MS/MS. The detection limit was found to be 0.4 fg for atrazine (m/z 174) and 0.2 fg for 2-chloro-4, 6-diamino-S-triazine (DACT) (m/z 129) (S/N = 3) in EESI/MS/MS. A linear dynamic range of 4–5 orders of magnitude (r = 0.996) was determined for both atrazine and DACT. A single sample analysis was completed using tandem EESI-MS/MS within 1 min, providing a practical convenient method for rapid analysis of trace amounts of targeted metabolites present in complex matrices. Thus, tandem EESI-MS is potentially useful for previously discovered biomarker detection in multiple applications such as clinical diagnosis, drug discovery and forensic science.     

Direct protein detection from biological media through electrospray-assisted laser desorption ionization/mass spectrometry

We report here using a novel technology-electrospray-assisted laser desorption ionization (ELDI)/mass spectrometry-for the rapid and sensitive detection of the major proteins that exist in dried biological fluids (e.g., blood, tears, saliva, serum), bacterial cultures, and tissues (e.g., porcine liver and heart) under ambient conditions. This technique required essentially no sample pretreatment. The proteins in the samples were desorbed using a pulsed nitrogen laser without the assistance of an organic matrix. The desorbed protein molecules were then post-ionized through their fusion into the charged solvent droplets produced from the electrospray of an acidic methanol solution; electrospray ionization (ESI) proceeded from the newly formed droplets to generate the ESI-like protein ions. This new ionization approach combines some of the features of electrospray ionization with those of matrix-assisted laser desorption ionization (MALDI), that is, sampling of a solid surface with spatial resolution, generating ESI-like mass spectra of the desorbed proteins, and operating under ambient conditions.     

Monitoring macromolecular complexes involved in the chaperonin-assisted protein folding cycle by mass spectrometry

We have used native mass spectrometry to analyze macromolecular complexes involved in the chaperonin-assisted refolding of gp23, the major capsid protein of bacteriophage T4. Adapting the instrumental methods allowed us to monitor all intermediate complexes involved in the chaperonin folding cycle. We found that GroEL can bind up to two unfolded gp23 substrate molecules. Notably, when GroEL is in complex with the cochaperonin gp31, it binds exclusively one gp23. We also demonstrated that the folding and assembly of gp23 into 336-kDa hexamers by GroEL-gp31 can be monitored directly by electrospray ionization mass spectrometry (ESI-MS). These data reinforce the great potential of ESI-MS as a technique to investigate structure-function relationships of protein assemblies in general and the chaperonin-protein folding machinery in particular. A major advantage of native mass spectrometry is that, given sufficient resolution, it allows the analysis at the picomole level of sensitivity of heterogeneous protein complexes with molecular masses up to several million daltons.     

Principles of electrospray ionization

Electrospray ionization is today the most widely used ionization technique in chemical and biochemical analysis. Interfaced with a mass spectrometer it allows the investigation of the molecular composition of liquid samples. With electrospray a large variety of chemical substances can be ionized. There is no limitation in mass which thus enables even the investigation of large noncovalent protein complexes. Its high ionization efficiency profoundly changed biomolecular sciences because proteins can be identified and quantified on trace amounts in a high throughput fashion. This review article focuses mainly on the exploration of the underlying ionization mechanism. Some ionization characteristics are discussed that are related to this mechanism. Typical spectra of peptides, proteins, and noncovalent complexes are shown and the quantitative character of spectra is highlighted. Finally the possibilities and limitations in measuring the association constant of bivalent noncovalent complexes are described.     

Ambient ionization mass spectrometry imaging for disease diagnosis: Excitements and challenges

Tissue analysis in histology is extremely important and also considered to be a gold standard to diagnose and prognosticate several diseases including cancer. Intraoperative evaluation of surgical margin of tumor also relies on frozen section histopathology, which is time consuming, challenging and often subjective. Recent development in the ambient ionization mass spectrometry imaging (MSI) technique has enabled us to simultaneously visualize hundreds to thousands of molecules (ion images) in the biopsy specimen, which are strikingly different and more powerful than the single optical tissue image analysis in conventional histopathology. This paper will highlight the emergence of the desorption electrospray ionization MSI (DESI-MSI) technique, which is label-free, requires minimal or no sample preparation and operates under ambient conditions. DESI-MSI can record ion images of lipid/metabolite distributions on biopsy specimens, providing a wealth of diagnostic information based on differential distributions of these molecular species in healthy and unhealthy tissues. Remarkable success of this technology in rapidly evaluating the cancer margin intraoperatively with very high accuracy also promises to bring this imaging technique from bench to bedside.     

Primary structure of a photoactive yellow protein from the phototrophic bacterium Ectothiorhodospira halophila,with evidence for the mass and the binding site of the chromophore.

The complete amino acid sequence of the 125-residue photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been determined to be MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKEVIGKNFFKDVAP+ ++ CTDSPEFYGKFKEGVASGNLNTMFEYTFDYQMTPTKVKVHMKKALSGDSYWVFVKRV. This is the first sequence to be reported for this class of proteins. There is no obvious sequence homology to any other protein, although the crystal structure, known at 2.4 A resolution (McRee, D.E., et al., 1989, Proc. Natl. Acad. Sci. USA 86, 6533-6537), indicates a relationship to the similarly sized fatty acid binding protein (FABP), a representative of a family of eukaryotic proteins that bind hydrophobic molecules. The amino acid sequence exhibits no greater similarity between PYP and FABP than for proteins chosen at random (8%). The photoactive yellow protein contains an unidentified chromophore that is bleached by light but recovers within a second. Here we demonstrate that the chromophore is bound covalently to Cys 69 instead of Lys 111 as deduced from the crystal structure analysis. The partially exposed side chains of Tyr 76, 94, and 118, plus Trp 119 appear to be arranged in a cluster and probably become more exposed due to a conformational change of the protein resulting from light-induced chromophore bleaching. The charged residues are not uniformly distributed on the protein surface but are arranged in positive and negative clusters on opposite sides of the protein. The exact chemical nature of the chromophore remains undetermined, but we here propose a possible structure based on precise mass analysis of a chromophore-binding peptide by electrospray ionization mass spectrometry and on the fact that the chromophore can be cleaved off the apoprotein upon reduction with a thiol reagent. The molecular mass of the chromophore, including an SH group, is 147.6 Da (+/- 0.5 Da); the cysteine residue to which it is bound is at sequence position 69.     

Direct measurement of islet amyloid polypeptide fibrillogenesis by mass spectrometry

A novel method for monitoring fibrillogenesis is developed and applied to the amyloidogenic peptide, islet amyloid polypeptide (IAPP). The approach, based on electrospray ionization mass spectrometry, is complementary to existing assays of fibril formation as it monitors directly the population of precursor rather than product molecules. We are able to monitor fiber formation in two modes: a quenched mode in which fibril formation is halted by dilution into denaturant and a real time mode in which fibril formation is conducted within the capillary of the electrospray source. Central to the method is the observation that fibrillar IAPP does not compromise the ionization of monomeric IAPP. Furthermore, under mild ionization conditions, fibrillar IAPP does not dissociate and contribute to the monomeric signal. Critically, we introduce an internal standard, rat IAPP, for analysis on the mass spectrometer. This standard is sufficiently similar in sequence in that it ionizes identically to human IAPP. Furthermore, the sequence is sufficiently different in that it does not form fibrils and is distinguishable on the basis of mass. Applied to IAPP fibrillogenesis, our technique reveals that precursor consumption in seeded reactions obeys first-order kinetics. Furthermore, a consistent level of monomer persists in both seeded and unseeded experiments after the fibril formation is complete. Given the inherent stability of fibrils, we expect this approach to be applicable to other amyloid systems.