VERMONT IMMUNOBIOL / INFECTIOUS DISEASES CTR: CORE C: PROTEOMICS ANALYSIS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 COBRE的蛋白质组学核心实验室具有Thermo-Finnigan LTQ线性四极杆离子阱液相色谱-质谱仪（LCMS）和Thermo-Finnigan LCQ Deca XP离子阱LCMS。 两种仪器都有电喷雾电离（ESI）作为主要入口，LTQ在纳米喷雾ESI中使用在芯中构建的入口进行操作，用于由熔融二氧化硅制成的微型LC柱，也用作纳米喷雾尖端。 这种方法将肽分析的检测限提高到飞摩尔范围。 过去一年的一个关键职能是马修斯博士继续与COBRE研究人员进行讨论，并与COBRE研究人员作为一个小组进行会谈，以说明核心的能力以及如何使用核心来解决COBRE研究人员的科学问题。 特别强调协助研究人员准备蛋白质组学分析的样品。 人员在核心和标准协议是在适当的地方进行蛋白质的肽的蛋白质组学分析LCMS，但获得样品到这一点是困难的部分。 随着COBRE研究人员开始了解蛋白质组学分析样品制备的性质，我们看到在分析提交的样品方面取得了更大的成功。 核心小组的另一个关键方面是与调查人员合作，分析他们的结果。 蛋白质组学实验由于数据溢出而导致死亡，而将数据减少到有意义的蛋白质鉴定术语或特定翻译后修饰的位置是困难的部分。 马修斯博士和他的工作人员在数据解释方面与研究人员密切合作。 质谱法蛋白质组学分析： 以下研究者提交了在2007年4月1日期间由核心测量和完成的样本  2008年3月31日： PI：加里沃德 教授 微生物学和分子遗传学 PI：Mariana Matrajt 助理教授 微生物学和分子遗传学 PI：Mercedes Rinc？n 副教授 医学  免疫生物学分部 PI：Terrence Delaney 副教授 植物生物学 PI：Jonathan Boyson 助理教授 手术 这些项目中的每一个都是由上述实验室的研究生和博士后研究员进行的。 个别学生或博士后的名字没有列出。 加里·沃德和玛丽安娜·马特拉伊特的项目集中在弓形虫上。Matrajt博士根据所完成的工作编写并提交了一份手稿。 Mercedes Rinc <$n的项目专注于通过胸腺细胞中的蛋白磷酸化进行细胞控制的细胞信号传导。 这项工作的成果是一份手稿，已被《科学》杂志接受。 Delaney博士的项目涉及拟南芥中的植物信号传导和植物对病原体的防御。 Boyson博士目前正在半不变NKT（iNKT）细胞中研究一种蛋白质复合物的亚单位。 各种不同的LCMS技术已被用于这些项目中，从鉴定被研究的生物体中尽可能多的蛋白质，以及生理和代谢条件的变化，到鉴定蛋白质修饰位点（例如蛋白质磷酸化位点鉴定）的重点研究。 双向凝胶电泳： 已组装了通过2D凝胶电泳分析和分离蛋白质的所有设备，包括BioRad ExQuest Spot Cutter和用于预分级溶液中样品的BioRad Microrotofor Cell。Ward博士和Haraldsen博士已经为Core中可用的2D凝胶方法编写了书面方案。我们在通过二维荧光差异凝胶电泳（2D-DIGE）分析样品方面积累了丰富的经验，目前正在评估新的Proteominer技术（BioRad），用于在二维凝胶分析之前富集低丰度蛋白质。 2007年1月4日期间  2008年3月31日，以下研究者使用了Core的2D凝胶功能： PI：加里沃德 教授 微生物学和分子遗传学 PI：Ralph Budd 教授 医学  免疫生物学分部 PI：Chris休士顿 助理教授 医学  传染病 PI：Jonathan Boyson 助理教授 手术 这些项目中的每一个都是由上述实验室的研究生和博士后研究员完成的;同样，没有列出个别学生或博士后的姓名。项目包括分析性2D凝胶电泳、2D-DIGE和蛋白质的制备分离，以通过LCMS进行鉴定。

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. The Proteomics Core Laboratory for the COBRE has both a Thermo-Finnigan LTQ linear quadrupole ion trap liquid chromatograph - mass spectrometer (LCMS) and a Thermo-Finnigan LCQ Deca XP ion trap LCMS. Both instruments have electrospray ionization (ESI) as the primary inlet, and the LTQ is operated in nanospray ESI using an inlet constructed in the Core for micro-LC columns made of fused silica that also function as the nanospray tip. This approach improves detection limits for peptide analysis into the femtomole range. A key function of the past year was continued discussion by Dr. Matthews with the individual COBRE investigators and as talks to the COBRE investigators as a group to relate the capabilities of the Core and how the Core could be used to address scientific problems of COBRE investigators. Special emphasis has been in assisting investigators in preparation of samples for proteomic analyses. Personnel is available in the Core and standard protocols are in place to perform digests of proteins to peptides for proteomic analysis by LCMS, but getting samples to that point is the difficult part. As COBRE investigators begin to understand the nature of the sample preparation for proteomic analysis, we are seeing better success in analyzing samples submitted. Another key aspect of the Core has been to work with investigators in analysis of their results. The proteomics experiments result in death by data overflow, and reducing data to meaningful terms of protein identification or location of specific posttranslational modifications is the hard part. Dr. Matthews and his staff have worked closely with investigators on data interpretation. Proteomics Analyses by Mass Spectrometry: The following investigators submitted samples that have been measured and completed by the Core during the period 4/1/2007  3/31/2008: PI: Gary Ward Professor Microbiology & Molecular Genetics PI: Mariana Matrajt Assistant Professor Microbiology & Molecular Genetics PI: Mercedes Rinc¿n Associate Professor Medicine  Immunobiology Div. PI: Terrence Delaney Associate Professor Plant Biology PI: Jonathan Boyson Assistant Professor Surgery Each of these projects was being performed by graduate students and postdoctoral fellows in the above laboratories. Individual student or postdoctoral names are not listed. The projects by Gary Ward and Mariana Matrajt focus on Toxoplasma gondii. One manuscript has been prepared and submitted by Dr. Matrajt from work performed. The project by Mercedes Rinc¿n focuses on cell signaling via protein phosphorylation in thymocytes for cellular control. This work has resulted in a manuscript that has been accepted by Science. The project of Dr. Delaney involves plant signaling and plant defense against pathogens in Arabidopsis thaliana. Dr. Boyson is looking to confirm identification of a sub-unit of a protein complex he is currently studying in semi-invariant NKT (iNKT) cells. A variety of different LCMS techniques have been used in these projects ranging from identification of as many proteins as possible in the organisms being studied and changes in physiologic and metabolic conditions to focused studies of identification of protein modification sites (e.g. protein phosphorylation site identification). Two-dimensional gel electrophoresis: All equipment for the analysis and isolation of proteins by 2D gel electrophoresis has been assembled, including the BioRad ExQuest Spot Cutter and a BioRad Microrotofor Cell for prefractionating samples in solution. Dr. Ward and Dr. Haraldsen have generated written protocols for the 2D gel methods available in the Core. We have developed extensive experience in the analysis of samples by 2D Fluorescence Difference Gel Electrophoresis (2D-DIGE) and we are currently evaluating the new Proteominer technology (BioRad) for the enrichment of low abundance proteins prior to 2D gel analysis. During the period 4/1/2007  3/31/2008, the following investigators made use of the 2D gel capabilities of the Core: PI: Gary Ward Professor Microbiology & Molecular Genetics PI: Ralph Budd Professor Medicine  Immunobiology Div. PI: Chris Huston Assistant Professor Medicine  Infectious Diseases PI: Jonathan Boyson Assistant Professor Surgery Each of these projects was performed by graduate students and postdoctoral fellows in the above laboratories; again, individual student or postdoctoral names are not listed. Projects have included analytical 2D gel electrophoresis, 2D-DIGE and preparative isolation of proteins for identification by LCMS.