DIGE Package: Variable Mode Imager and Spot Picker

描述（由申请人提供）：我们已经确定了几个NIH资助的调查员和两个多机构核心设施，他们的研究项目涉及研究蛋白质表达变化与异常细胞和/或有机体生理学的关系。此应用程序中包含的主要用户项目涵盖各种学科，包括：血液学、风湿病学、艾滋病研究、心脏病学、肿瘤学和细胞生物学。我们需要一个集成系统，包括激光扫描仪（台风三重奏），点切割机（伊坦点拾取器），驱动/分析软件和必要的计算。该集成系统将使用户能够使用2d凝胶进行蛋白质表达的比较分析，量化差异表达的大小，并分离和鉴定存在于差异“点”中的蛋白质-后者通过与TSRI现有的LC-MS能力相结合而成为可能。这种共享设备的主要新功能是能够对多种蛋白质样品进行DIGE分析，如下所述。DIGE（差异凝胶分析）是一种更准确，可重复性和更少麻烦的方法，用于重复样品的比较蛋白质组分析。该系统将被安置在现有的NCI资助的蛋白质组学核心中，该核心位于TSRI分子和实验医学系（MEM）。目的：本提案的长期目标是为TSRI和邻近机构的研究人员提供可靠，可重复和定量的比较蛋白质组学研究方法。具体目标：在MEM蛋白质组学核心中建立全面的DIGE服务。我们将购买一台“台风”可变模式成像仪和“伊坦”定点采集器，并将这些设备与现有服务集成，以提供全面的DIGE能力。用户将决定其项目所需的核心支持的程度。全面的核心服务将包括：用菁荧光团标记蛋白质样品，二维电泳，台风图像捕获和使用Decyder软件进行数据分析；利用Ettan摘点器生成摘点清单并进行切割；蛋白质斑点的处理，包括酶切、LC-MS和MS谱的蛋白质数据库查询。将提供一台专用计算机工作站，使用Decyder和Decyder扩展数据分析软件进行数据分析。它还将作为新用户学习软件应用程序的培训站，并可以访问分析软件的在线教程。第二个“浮动”许可证将用于软件，允许不在设施附近的团体进行远程数据分析。相关性：确定的用户群体都参与了与人类健康高度相关的特定疾病研究，并得到了几个NIH研究所的支持。比较蛋白质组学研究，如本申请中概述的那些，是对基因表达阵列数据的补充，但提供了额外的见解，了解基因活性的变化如何通过改变蛋白质丰度、蛋白质同工型和蛋白质修饰转化为疾病表型。

DESCRIPTION (provided by applicant): We have identified several NIH funded Investigators and two multi-institution core facilities whose research projects involve investigation of how changes in protein expression relate to abnormal cellular and/or organismal physiology. Major user projects included in this application cover a variety of disciplines, including: hematology, rheumatology, AIDS research, cardiology, oncology and cell biology. We are requesting an integrated system including a laser scanner (Typhoon Trio), spot cutter (Ettan Spot Picker), driver/analysis software and necessary computing. This integrated system will enable users to perform comparative analysis of protein expression using 2-D gels, to quantify the magnitude of differential expression, and to isolate and identify proteins present in differential 'spots'-the latter made possible by coupling with existing LC-MS capability at TSRI. The major new capability this shared equipment confers is the ability to multiplex protein samples to perform DIGE analysis as described below. DIGE (difference gel analysis) is a more accurate, reproducible and less cumbersome method for comparative proteome analysis of replicate samples. This system will be housed in an existing, NCI funded Proteomics Core located in the Department of Molecular and Experimental Medicine (MEM) at TSRI. Objective: The long-term objective of this proposal is to provide researchers at TSRI and neighboring institutions a reliable, reproducible and quantitative method for comparative proteomic studies. Specific Aim: Establish Comprehensive DIGE service in the MEM Proteomics Core. We will purchase a Typhoon variable mode imager and Ettan spot picker and integrate this equipment with existing services to provide a comprehensive DIGE capability. Users will determine the extent of core support required for their project. Comprehensive core services will include: labeling protein samples with cyanine fluorophores, 2-D electrophoresis, image capture on Typhoon and data analysis using Decyder software; generation of spot pick list and cutting using Ettan spot picker; and processing of protein spots including digestion, LC-MS and protein database queries of MS profiles. One dedicated computer workstation will be provided for data analysis using Decyder and Decyder extended data analysis software. It will also serve as a training station for new users learning software applications and will have access to online tutorials for analysis software. A second 'floating' license will be maintained for software to allow remote data analysis by groups not located near the facility. Relevance: Identified user groups are all involved in disease specific research that is highly relevant to human health, and receive support from several NIH institutes. Comparative proteomic studies such as those outlined in this application are complementary to gene expression array data, but provide additional insight into how changes in gene activity translate into disease phenotypes by altering protein abundance, protein isoforms and protein modifications.