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2010全国质谱大会大会报告(四)

2010.8.05

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辉瑞-惠氏(Pfizer/Wyeth) Feng, Xidong(冯喜东)教授

  来自辉瑞-惠氏(Pfizer/Wyeth)的Feng, Xidong(冯喜东)教授做了题为“Structure Elucidation of Natural Product Cyclic Peptides with Labile Side-Chain Modifications Using CID, IRMPD and ECD FTMS/MS(基于CID, IRMPD 和ECD FTMS/MS的侧链改性研究天然产物环肽的结构鉴定)”的报告。

  冯教授在报告中介绍了,通过Multi-CHEF、SORI-CID、iRMPO和ECO实验以及基因交替的方法,使用FTMS/MS,用不稳定的侧链改性的方法阐明未知天然产物环肽的结构。结果表明,具有ppm检测精度的FTMS适合于研究不常见的氨基酸残基的序列。

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加拿大Alberta大学实验室医学和病理学系分析和环境毒理室 Li,Xing-Fang教授

  来自加拿大Alberta大学实验室医学和病理学系分析和环境毒理室的Li,Xing-Fang教授,做了题为“Mass Spectrometry Techniques for Discovery of Carcinogens in Drinking Water and Studies of Health Effects”(用质谱技术发现饮用水中的致癌原和对健康影响的研究)的报告。在报告中,李教授首先介绍了饮用水污染消毒中现存的一些问题。饮用水的微生物污染仍是全球范围内水引起的疾病的重要原因,而饮用水消毒中会引入消毒副产物(DBPs),是消毒剂(如氯和氯胺)和水中的天然有机物(如NOM)反应产生的。为了控制引用水质,在很多国家,如三卤代甲烷(THMs)和卤代乙酸(HAAs)的消毒副产物已被替代。流行病学的研究表明,饮用氯代的水会增加患膀胱癌的风险。然而,从过去30年积累的证据表明,已被调节的DBPs不是膀胱癌风险增加的原因,或在生殖研究中观察到其反作用。还有其它不明物毒性更大,会在更低水平上生成DBPs。为了应对饮用水安全在分析和生物分析方面的挑战,课题组发展了一系列液相色谱-串联质谱技术用于分析饮用水中超痕量的致病原。课题组发现了一些以前未知的DBPs,可能是膀胱癌的致病原,比如饮用水中的亚硝胺类、卤代苯醌、氯代phanazines。该报告举例了用串联质谱法对DBP-DNA结合的研究,可作为基因毒性测试的工具,有助于在立法中考虑DBPs的优先级。以下是英文摘要:

  Microbial contamination of drinking water is still the major cause of water-borne disease affecting millions of people around the world. Disinfection of drinking water is the most effective public health measure to disinfect pathogens to eradicate water-borne diseases. However, disinfection of water unintentionally results in formation of disinfection byproducts (DBPs) from the reactions between disinfectants (e.g. chlorine and chloramines) and natural organic matters (NOM) in water. In order to control drinking water quality, surrogate DBPs such as several trihalomethanes (THMs) and haloacetic acids (HAAS) are currently regulated in the most countries. Epidemiological studies have shown potential association of drinking chlorinated water and increased risk of developing bladder cancer. However, accumulating evidence from past 30 years suggests that the regulated DBPs are not the cause of the increased bladder cancer risk or adverse reproductive effects that have been observed in population studies. Other unidentified, yet more toxic, DBPs may be produced at much lower levels. To address the analytical and bioanalytical challenges in drinking water safety, we have developed a set of liquid chromatography and tandem mass spectrometry techniques for ultra sensitive detection of carcinogens in water. We have discovered some previously unknown DBPs that are likely bladder carcinogens, such as nitrosamines, haloquinones, and chloro-phanazines in drinking water. In the present study, we report tandem mass spectrometry study of DBP-DNA binding as a tool for genotoxicity testing to assist prioritization of DBPs for regulatory consideration.

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美国普渡大学癌症研究中心 Tao,Andy(陶纬国)教授

  来自美国普渡大学癌症研究中心的Tao,Andy(陶纬国)教授,做了题为“In-Depth Phosphoproteome Analyses Using PolyMAC”(用PolyMAC深入分析磷酸化蛋白质组)的报告。在报告中,陶教授介绍了课题组发展的一种磷酸化肽的富集方法,称为PolyMAC(基于聚合物的金属离子亲和富集)。该试剂的核心部分是水溶性并容易从聚合物中获得的。课题组对PolyMAC和TiO2方法在专一性、重现性和灵敏度方面进行了比较;并比较了用几种金属离子功能化的PolyMAC试剂。富集后的磷酸化肽用纳升级液相色谱-串联质谱联用(nLC-MS/MS)方法分析。液相为Eksigent公司的ultra 2D LC系统,质谱用LTQ Orbitrap velos,数据用Proteome Discovery软件的SEQUEST方法检索。结果显示,仅使用50 ug的溶胞物,一次实验,PolyMAC富集后即可鉴定1000个磷酸化肽,具有95%以上的选择性。总体上,PolyMAC方法显示了优秀的选择性,出众的回收率和高重现性,是今日最有效的磷酸化肽富集技术。课题组还利用该方法研究了乳腺癌体系。以下是英文摘要:

  Protein phosphorylation plays critical roles in the regulation of many cellular functions. Analysis of phosphoproteomes by mass spectrometry depends on an efficient method to enrich phosphopeptides from complex mixtures. The current prevailing enrichment methods lack required efficiency and reproducibility due to complex sample conditions. Here, we utilize a novel soluble nanopolymer-based phosphopeptide enrichment approach termed PolyMAC (Polymer-based Metal ion Affinity Capture) for in-depth phosphoproteome analysis.

  The central piece of PolyMAC reagents lies in a water soluble and highly accessible polymer support. PolyMAC reagents are synthesized from polyamidoamine (PAMAM) dendrimers functionalized with metal ions to specifically capture O-phosphorylated peptides through bidentate chelation chemistry. An extensive comparison was made between PolyMAC reagents and the TiO2 method, in terms of the specificity, reproducibility and sensitivity. We also compared different types of PolyMAC reagents functionalized with several metal ions. The enriched phosphopeptides were analyzed by nanoflow liquid chromatography tandem mass spectrometry (Nlc-MS/MS). The MS spectra were acquired on an Eksigent ultra 2D LC system that is coupled to hybrid liner ion trap orbitrap mass spectrometer (LTQ Orbitrap velos, Thermo Fisher). Data were searched using the SEQUESTTM algorithm within the Proteome Discoverer software. Using only 50 ug of total cell lysate, PolyMAC enrichment has led to the identification of over 1000 phosphopeptides with over 95% selectivity in a single run. Overall, the PolyMAC method demonstrated excellent selectivity, outstanding recovery yield and high reproducibility, thus rendering it one of the most effective phosphopeptide isolation techniques to date.

  The PolyMAC enrichment approach has been applied to examine the differences in signaling pathways in breast cancer cell lines modulated by a tumor suppressor,spleen tyrosine kinase (Syk). MDA-MB-231 human breast cancer cell line was transfected with a tetracycline-inducible Syk vector. Proteins from cells with or without Syk induction were processed and phosphotyrosine peptides were enriched using the combination of pTyr peptide immunoprecipitation and PolyMAC to obtain the phosphorylation profiles. We identified 794 sites of tyrosine phosphorylation in malignant breast cancer cells,514 of which are dependent on the expression of Syk. Proteins with changes in pTyr phosphorylation were manually validated and a number of them confirmed through immunoprecipitation-Western blot experiments. They were mapped in a variety of major signaling networks including cell migration and apoptosis, therefore offering numerous leads to future breast cancer studies.

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清华大学 林金明教授

  来自清华大学的林金明教授做了题为“纸基电喷雾质谱常态离子源直接测定复杂样品”的报告。质谱是测定复杂样品的有效手段,能够提供待测物的分子信息。目前的质谱分析通常需要对复杂样品进行预处理和预分离,无法实现快速和高效分析。为解决复杂样品直接检测过程中的样品前处理难题,林教授的课题组提出了一种称为纸基电喷雾的新型常态离子源。将样品直接加载到三角形的色谱纸上,接通高压电后即可直接生成电喷雾,能够进行直接的质谱检测。

  与Nano-ESI的对比实验表明,纸基电喷雾比Nano-ESI能保留更多的分子离子,其电离能力更软。计算表明,纸基电喷雾产生的分子离子具有的内能比Nano-ESI产生的分子离子内能低0.4电子伏左右,因此更适合生物分子样品的质谱分析。

  这一离子源能够适用于大多数的化合物,包括小分子有机物、肽和蛋白质等。利用在纸上进行液体干燥后检测的方法还可以直接测定血样和尿样中的各种药物和代谢物。利用纸的采样能力能够直接分析固相表面的样品,测定快速,灵敏度高。将纸基电喷雾与便携式质谱仪联用,能够拓宽在非实验室环境下的复杂样品检测能力。最后,林教授指出,纸基电喷雾具体的机理还有待于进一步的实验证明。

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