MALDI & ESI & LC ESI QOTOF INSTRUMENT & METHOD DEVELOPMENT

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。我们的实验室广泛使用两台应用生物系统API QSTAR(Tm)Pulsar I QqTOF质谱仪来表征从合作研究项目中获得的复杂生物样品。与ABI(马萨诸塞州弗雷明翰)和Sciex(On.在加拿大)，我们继续调查这些仪器的性能，并继续与ABI合作开发该仪器的软件和数据分析。目前，我们的重点是建立用于蛋白质组和糖组学应用的常规毛细管LC-IDA-MS/MS，以及对提交给MS资源的大量生物样本进行表征。针对典型的蛋白质消化、脂类和碳水化合物，IDA MS/MS自动化功能的使用正在不断优化，并生成了批处理参数文件，以便于实验室成员使用。处理脚本已经过优化，可以在用于数据分析的工作站上使用。2004年安装了Waters CapLC；灵敏度可以很好，但频繁的管道问题干扰了直径小于100微米的柱的高吞吐量使用。使用较小直径的柱组件可显著提高灵敏度，从而能够分析低浓度或限量的样品。因此，组合系统的最佳运行应该为重要生物样品的分析提供一种替代的、改进的方法。该项目目前的阶段可以简单地分为两个主要目标；首先是优化Waters CapLC系统，然后是方法开发研究，以补充在MS资源范围内进行的当前研究。在大量的仪器优化之后，目前的方法开发正在进行以蛋白质组学为基础的研究，包括磷酸化分析。未来的方法开发方向包括分析其他翻译后修饰，包括糖基化和碳水化合物样本。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our laboratory makes extensive use of two Applied Biosystems API QSTAR(tm) Pulsar i QqTOF mass spectrometers for the characterization of complex biological samples obtained from collaborative research projects. In agreement with ABI (Framingham, MA) and Sciex (ON. Canada), we continue to investigate the performance of these instruments and we continue to work with ABI on software development for this instrument and data analysis. Currently, our focus is on establishing routine capillary LC-IDA-MS/MS for proteomics and glycomics applications, in addition to characterization the large variety of biological samples that are submitted to the MS Resource. Use of automated IDA MS/MS functions are continuously being optimized for typical protein digests, lipids and carbohydrates, and batch parameter files have been generated for ease of use by the members within the laboratory. Processing scripts have been optimized for use on workstations for data analysis. A Waters capLC was installed in 2004; sensitivity can be excellent but frequent plumbing problems have interfered with high throughput use of columns having diameters less than 100 um. The use of the smaller diameter column assemblies offers a significant increase in sensitivity, enabling samples of low concentrations or limited quantity to be analyzed. Consequently, the optimum operation of the combined system should offer an alternative, improved approach for analysis of important biological samples. The current phase of this project can be simply divided into two principal aims; primarily the optimization of the Waters CapLC system, followed by method development studies to complement the current research undertaken within the MS resource. Following substantial instrument optimization, current method development is being pursued for proteomics-based studies, including phosphorylation analysis. Future directions for method development include the analysis of other post-translational modifications, including glycosylation, and carbohydrate samples.