LC ESI QOTOF METHOD DEVELOPMENT AND NEW INSTRUMENTATION EVALUATION

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 我们的实验室使用两台Applied Biosystems API QSTAR（tm）Pulsar i QqTOF质谱仪和一台配备沃茨Acuity LC的Agilent chip-LC Q-TOF质谱仪，通过ESI和MALDI对从合作研究项目中获得的复杂生物样品进行表征。目前，我们的重点是建立常规的毛细管LC-IDA-MS/MS，用于蛋白质组学、糖组学和脂质组学应用，以及对提交给MS资源的各种生物样品进行表征。自动化IDA MS/MS功能的使用正在针对典型的蛋白质代谢物、脂质和碳水化合物进行不断优化，并且已经生成了批参数文件，以便于实验室内的成员使用。处理脚本已经过优化，可在工作站上用于数据分析。使用较小直径的色谱柱组件可显著提高灵敏度，从而能够分析低浓度或有限数量的样品。LC系统的优化之后是方法开发研究，以补充MS资源内进行的当前研究。正在为糖组学、脂质组学和蛋白质组学研究开发方法，包括磷酸化分析。正在探索使用在线ESI-MS分析TLC分离的样品。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Our laboratory uses two Applied Biosystems API QSTAR(tm) Pulsar i QqTOF mass spectrometers with Waters Acuity LCs and an Agilent chip-LC Q-TOF mass spectrometer for the characterization of complex biological samples obtained from collaborative research projects by ESI and MALDI. Currently, our focus is on establishing routine capillary LC-IDA-MS/MS for proteomics, glycomics and lipidomics applications, in addition to characterization of 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. 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. Optimization of the LC systems is followed by method development studies to complement the current research undertaken within the MS resource. Method development is being pursued for glycomics, lipidomics and proteomics-based studies, including phosphorylation analysis. The use of online ESI-MS for analysis of TLC-separated samples is being explored.