Proteomics Core

蛋白质组学核心(核心B)提供最先进的分析仪器和专业知识 在这个PPG中向调查人员介绍蛋白质组学。蛋白质组学核心的一个独特功能是所有 主要蛋白质组学分析技术，在分析方法上提供了非凡的灵活性。这个 蛋白质组学核心提供1)简单和复杂多蛋白样本中的蛋白质鉴定， 包括多蛋白复合体的蛋白质组分，2)差异蛋白质的定量分析 表达和修饰；3)蛋白质修饰的分析。 蛋白质组学核心员工提供实验设计和样品制备方面的咨询，以及 消化、蛋白质和多肽分离、MS分析和初级数据处理。两者都是电喷雾 用于LC-MS-MS实验的电离(ESI)质谱仪和MALDI TOF-TOF仪器 可用于MS分析。DGE/MS技术包括荧光染料和复合2D凝胶 直接定量比较的内部标准。自动化工作站用于蛋白质切除 和样品处理，然后是MALDI-TOF MS和TOF/TOF MS/MS和自动化数据库 用Mascot软件进行蛋白质鉴定。Core还提供与N终端的相对量化 反应性差示标记试剂(iTRAQ和同位素标记的苯异氰酸酯) 修饰蛋白质形式的定量。为了从多蛋白复合体中识别蛋白质， 多维LC直接与自动LC-MS-MS联用，然后是自动数据库 使用SEQUEST和Mascot软件进行搜索。SEQUEST搜索是在高速多处理器上完成的 高级计算研究和教育中心(ACCRE)中的Linux集群。基于SEQUEST 使用开源工具(多肽/蛋白质预言家)进行评估和筛选，并进行集成 具有定制设计的数据库系统(芯片)，用于组织、筛选和比较数据集。 SALSA和P-Mod的附加数据分析能够识别翻译后修改， MS-MS数据的加合物或序列变体。 项目1(PEEK)将使用DGE/MS差异显示技术，项目3(封面)将 利用简单和复杂多蛋白质样品的蛋白质鉴定技术。项目2 (Polk)将大量利用这两项服务。对蛋白质修饰的分析在修订后的 项目，但可用于后续/补充研究。

The Proteomics Core (Core B) provides state-of-the-art instrumentation and expertise in analytical proteomics to investigators in this PPG. A unique feature of the Proteomics Core is the availability of all major proteomics analysis technologies, which offers exceptional flexibility in analytical approaches. The Proteomics Core provides 1) identification of proteins in both simple and complex multiprotein samples, ncluding protein components of multiprotein complexes, 2) quantitative analyses of differential protein expression and modification, and 3) analyses of protein modifications. Proteomics Core staff provide consultation on experimental design and sample preparation, as well as digestion, protein and peptide separations, MS analyses and primary data workup. Both electrospray onization (ESI) mass spectrometers for LC-MS-MS experiments and MALDI TOF-TOF instruments are available for MS analyses. DIGE/MS technology includes fluorescent dyes and multiplexed 2D gels with nternal standards for direct quantitative comparisons. Automated workstations are used for protein excision and sample processing, followed by MALDI-TOF MS and TOF/TOF MS/MS and automated database searching with Mascot software for protein identification. The Core also offers relative quantitation with Nterminal reactive differential labeling reagents (iTRAQ and isotopically-labeled phenylisocyanate) for relative quantitation of modified protein forms. For protein identification from multiprotein complexes, multidimensional LC is coupled directly with automated LC-MS-MS, followed by automated database searching with Sequest and Mascot software. Sequest searches are done on a high speed, multiprocessor Linux cluster in the Advanced Computing Center for Research and Education (ACCRE). Sequest-based identifications are evaluated and filtered using open-source tools (Peptide/Protein Prophet) and integrated with a custom-designed database system (CHIPS) for organizing, filtering and comparing data sets. Additional data analysis by SALSA and P-Mod enables identification of posttranslational modifications, adducts or sequence variants from MS-MS data. Project 1 (Peek) will utilize the DIGE/MS differential-display technology, and Project 3 (Cover) will utilize the protein identification technologies from both simple and complex multiprotein samples. Project 2 (Polk) will heavily utilize both services. Analysis of protein modifications is no longer proposed in the revised Projects, but may be used for subsequent/complementary studies.