MS FOR HIGH THROUGHPUT PROTEIN IDENTIFICATION

12个主要用户申请资金，建立一个中央集成蛋白质鉴定系统，允许高通量分析2D凝胶中的蛋白质斑点。所要求的仪器包括质谱仪、二维凝胶系统和相关附件。这项技术是在过去五年里在西雅图华盛顿大学约翰·R·耶茨三世博士的实验室里开发的。将感兴趣蛋白质点的实验确定的串联质谱图(MS/MS)与现有序列数据库中存在的相应大小的每个可能的肽的计算机生成的理论串联质谱图进行比较。这项技术在基因组已经完全测序或有大型EST数据库的生物体中使用时特别成功。也有可能识别物种间保守的蛋白质区域，或者使用相同的仪器从MS/MS光谱中进行直接测序。因此，质谱学将提供一种极其强大和快速的方法来确定感兴趣的蛋白质的序列，并将其识别为已知或未知，从而为后续实验确定其功能奠定基础。这12名研究人员将利用这一仪器来研究各种生物学问题，这将加强他们由NIH资助的研究。此外，这种仪器将促进健康科学中心许多其他研究人员的研究，他们只需要鉴定几种蛋白质。健康科学中心目前还没有类似的仪器可用。这一仪器将由癌症中心、院长办公室和一个部门联盟出资购买的其他新的蛋白质化学设备来补充。这个SGI上的小组有合作互动的历史，这一背景很好地证明了所请求的仪器的成功共享。

Twelve major users request funds to establish a central integrated protein identification system which allows high-throughput analysis of protein spots from 2D gels. The instrumentation requested consists of a mass spectrometer, 2D gel system and associated accessories. This technology has been developed during the last five years in the laboratory of Dr. John R. Yates, III, at the University of Washington, Seattle. The experimentally determined, tandem mass spectrum (MS/MS) for a protein spot of interest is compared with the compute generated, theoretical tandem mass spectra for every possible peptide of the corresponding size present within existing sequence databases. This technique is particularly successful when used in an organism whose genome has been completely sequenced, or one for which a large EST database is available. It is also possible to identify regions of proteins that are conserved between species, or to perform direct sequencing from MS/MS spectra using the same instrumentation. Thus, mass- spectrometry will provide an extremely powerful and rapid method for determining the sequences of interesting proteins and identifying them as known or unknown, thereby forming the foundation for subsequent experiments to determine their function. The twelve investigators will make use of this instrumentation to pursue various biological questions which will enhance their NIH funded research. In addition this instrumentation will facilitate the research of many other investigators in the Health Sciences Center who require identification of only a few proteins. No comparable instrument is currently available at the Health Sciences Center. This instrumentation will be compliment by other new protein chemistry equipment purchased with funds from the Cancer Center, Dean's Office and a consortium of Departments. The group on this SGI has a history of collegial interactions, a background that argues well for the successful sharing of the instrumentation requested.