2010全国质谱大会大会报告(四)

2010-8-05 17:37 来源: 分析测试百科
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中国科学院大连化学物理研究所 许国旺研究员

  来自中国科学院大连化学物理研究所的许国旺研究员做了题为“液相色谱-质谱用于代谢标志物的发现和鉴定”的报告。许老师提出,现在是一个组学的时代,代谢组学作为最主要的组学之一,目前在疾病研究、药物研发及职务和微生物等领域均得到重视。

  代谢组学是研究小分子(相对分子质量小于1, 000)一个十分有用的工具,它以组织、体液或细胞为研究对象。一般来说,代谢组学的研究包括样品采集、预处理、代谢组数据采集、多变量数据分析、标志物发现和识别及最终的生物解释几方面。由于缺乏数据库样本及标样,代谢标志物的识别是研究的一个瓶颈。

  许老师在报告中,讲述了基于色谱-质谱联用技术的集成识别策略,包括多变量数据分析、精确分子量测定、质谱碎片裂解规律、色谱保留规律、馏分微制备、亲和色谱、酶解等。并列举了药物作用机理和疾病标志物的代谢组学研究作为实例阐述了这一集成策略。

Wisconsin-Madison(威斯康星-麦迪逊)大学 Li,Lingjun(李灵军)教授

  来自Wisconsin-Madison(威斯康星-麦迪逊)大学的Li,Lingjun(李灵军)教授,做了题为“Probing Neuronal Communication via Novel Mass Spectral Strategies”(用新型质谱技术探索神经通讯)的报告。她首先介绍了神经肽在细胞间通讯中至关重要的作用,这些聚合多肽在化学信使调节,和在神经电路中测定它们的功能中具有重要作用。甲壳动物具有较简单和已被充分研究的神经系统,可作为很好的模型系统来发展分析方法,并观测神经肽丰富的全部细节是如何来精细调节神经电路,并在细胞和神经网络水平产生多种输出。使用高灵敏度的基于质谱的多肽图和从头测序方法,发现了大量的新型多肽,并发现即使一个相对简单的神经元网络,同样包含了未被预期的丰富多样的神经肽。另外,质谱成像技术和活体微透析取样工具,可史无前例地对神经肽分布和分泌的细节进行追踪。对于完成对生物活性的神经肽的功能发现的目标,发展了建立在同位素甲醛标记和多同位素标记(N,N-二甲基亮氨酸)的新型定量技术基础上的方法,可对不同生理条件下的神经肽组进行区分显示。李教授介绍了神经肽在摄食行为和环境压力下的调节。总而言之,这种多肽组学结合生理学的研究,有助于阐释在调节神经网络可塑性方面,神经肽扮演的功能。以下是英文摘要:

  Neuropeptides make up the largest and the most complex signaling molecules used in intercellular communication. Because of critical roles that these polypeptides play in the regulation of chemical messengers and determine their functions in the neural circuitry. The simpler and well-characterized crustacean nervous system provides an excellent model system to facilitate analytical method development and to investigate how a rich repertoire of neuropeptides can fine tune a well-defined neural circuit that produces multiple outputs at the cellular and network levels. Using a highly sensitive mass spectrometry-based peptide profiling and de novo sequencing strategy, a large number of novel peptides have been discovered, revealing that even a relatively simple neural network contains an unexpectedly-rich diversity of neuropeptides. Furthermore, both mass spectrometric imaging techniques and in vivo microdialysis sampling tools have been implemented to follow neuropeptide distribution and secretion in unprecedented details. Towards the goal of functional discovery of bioactive neuropeptides, novel quantitative schemes based on isotopic formaldehyde labeling and multiplexed isobaric labeling based on N.N-dimethylated leucine have been developed to produce differential display of neuropeptidomes under different physiological. Example of neuropeptide regulation of feeding behavior and environmental stress will be described in this presentation. Collectively, these combined peptidomic and physiological studies will help to elucidate the function roles that neuropeptides play in regulating neural network plasticity.

加拿大Alberta大学 Le,Chris(乐晓春)教授

  来自加拿大Alberta大学的Le,Chris(乐晓春)教授,做了题为“Mass Spectromety and Affinty Chromatography Techniques for Studying Arsenic-binding Proteins in Human Cells”(在研究人类细胞中砷结合蛋白中的质谱和亲和色谱技术)的报告。砷中毒对于疾病、环境等的影响众所周知,但砷中毒的机理还不清楚,可能是三价砷结合了蛋白的硫基团,因此改变了蛋白的构象抑制了蛋白功能。为研究与蛋白作用的砷,课题组开发了一种亲和选择技术并与串联质谱联用,从大量的细胞蛋白中选择和鉴定特定的与砷结合的蛋白。受控实验使用包含游离半胱氨酸或非活性半胱氨酸的蛋白,显示砷亲和柱特异捕获游离的半胱氨酸。该方法被应用到牛胆绿素还原酶B、A549人肺癌细胞亚细胞器组分等研究中。使用亲和色谱-串联质谱方法鉴定大量的砷结合蛋白,因为其重要的生物功能引起科学家的关注。实验证实砷可以结合细胞提取物的蛋白质,而砷如何影响生物系统中的蛋白功能,需要继续研究活体细胞中砷和蛋白的相互作用才能确认。以下是英文摘要:

   Arsenic is one of the most important environmental agents in causing chronic human disease. Elevated levels of arsenic drinking water may affect >100 million people around the world .A wide variety of adverse health effects, most seriously, cancers of bladder, lung, urinary tract, and skin, have been attributed to chronic exposure to arsenic . However, the biochemical mechanisms responsible for these effects caused by arsenic remain unclear ,but may be mediated by the binding of trivalent arsenicals to thiol groups in proteins, thereby changing the conformation of these proteins and inhibiting their functions. If some of the affected proteins are responsible for cellular repair of DNA damage, for example, the inhibition of these proteins could lead to carcinogenesis.

   To study interaction of arsenic with proteins, we have developed an affinity selection technique, coupled with mass spectrometry, to select and identify specific arsenic-binding proteins from a large pool of cellular proteins. Controlled experiments using proteins either containing free cysteine(s) or inactive cysteine showed that the arsenic affinity column specifically captured the proteins containing free cysteine(s) available to bind tu arsenic .The technique was able to capture and identify trace amounts of bovine biliverdin reductase B present as a minor impurty in the commercial preparation of carbonic anhydrase II ,demonstrate the ability to identify arsenic-binding proteins in the presence of a large excess of non-specific proteins. application of the technique to the analysis of subcellular fractions of A549 human lung carcinoma cells identified 50 proteins in the analysis of subcellular fraction ,and 24 proteins in the membrane/ organelle fraction that could bind to arsenic .This added substantially to the current list of only a few known arsenic-binding proteins.

   A mumber of arsenic-binding proteins identified using the affinity chromatography tandem mass spectrometry approach were of particular interest because of their important biological functions .For example ,DNA-dependent protein kinase, ATP-dependent helicase II (Ku 70),and topoismerase 2 alpha , are involved in DNA repair and maintaining genome stability .several other proteins modulate the redox status of cells , e.g . peroxiredoxin-1 and thioredoxin ,and apoptosis ,e.g.,lamin A and heat shock cognate protein .this work shows that arsenic can bind to these proteins in cells extracts。How arsenic affects the function of these proteins in biological systems will have to be confirmed by studying arsenic interaction with proteins in living cells.