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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 Cobre的蛋白质组学核心实验室既有Thermo-Finnigan LTQ线性四极离子陷阱液相色谱-质谱仪(LCMS)，也有Thermo-Finnigan LCQ Deca XP离子陷阱LC MS。这两种仪器都有电喷雾电离(ESI)作为主要入口，LTQ在纳米喷雾电离中操作，使用由熔融二氧化硅制成的微型LC柱的核心中构建的入口，该入口也用作纳米喷雾尖端。这种方法将多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调查人员个人进行讨论，并作为一个小组与Cobre调查人员交谈，讲述核心的能力以及如何利用核心来解决Cobre调查人员的科学问题。特别强调协助研究人员准备用于蛋白质组分析的样本。核心中心有工作人员，也有标准的方案进行蛋白质到多肽的消化，以供LC MS进行蛋白质组学分析，但要做到这一点，获取样本是困难的部分。随着Cobre研究人员开始了解蛋白质组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。蛋白质组学实验通过数据溢出导致死亡，而将数据简化为有意义的蛋白质识别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一直在数据解释方面与调查人员密切合作。 蛋白质组学的质谱学分析 下列调查员提交了核心在2007年4月1日和2008年3月31日期间测量和完成的样本： 派：加里·沃德 微生物学与分子遗传学教授 少年派：玛丽安娜·马特拉伊特 微生物学与分子遗传学助理教授 圆周率：梅赛德斯·林肯 医学免疫生物学系副教授。 少年派：特伦斯·德莱尼 植物生物学副教授 少年派：乔纳森·博伊森 助理教授外科学 每个项目都是由上述实验室的研究生和博士后研究员执行的。个别学生或博士后的名字没有列出。 加里·沃德和玛丽安娜·马特拉伊特的项目重点是弓形虫。Matrajt博士已经准备并提交了一份手稿。梅赛德斯·林恩的这个项目专注于通过胸腺细胞中蛋白质磷酸化的细胞信号来进行细胞控制。这项工作的结果是一份已被《科学》杂志接受的手稿。德莱尼博士的项目涉及拟南芥中的植物信号和植物防御病原体。博伊森博士希望确认他目前正在半不变NKT(INKT)细胞中研究的蛋白质复合体的一个亚单位的鉴定。在这些项目中使用了各种不同的LC MS技术，从识别被研究生物中尽可能多的蛋白质和生理和代谢条件的变化到识别蛋白质修饰位点的重点研究(例如，蛋白质磷酸化位点识别)。 双向凝胶电泳法： 通过2D凝胶电泳法分析和分离蛋白质的所有设备已经组装完成，包括BioRad ExQuest Spot Cutter和用于在溶液中预分离样品的BioRad Microrootofor Cell。Ward博士和Haraldsen博士已经为Core中提供的2D凝胶方法制定了书面方案。我们在2D荧光差示凝胶电泳法(2D-DGE)样品分析方面积累了丰富的经验，目前我们正在评估新的Proteominer技术(BioRad)，用于在2D凝胶分析之前对低丰度蛋白质进行浓缩。 在2007年4月1日至2008年3月31日期间，下列调查员利用了核心的二维凝胶能力： 派：加里·沃德 微生物学与分子遗传学教授 少年派：拉尔夫·巴德 医学免疫生物学系教授。 少年派：克里斯·休斯顿 医学传染病学助理教授 少年派：乔纳森·博伊森 助理教授外科学 每个项目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名字。项目包括分析性2D凝胶电泳法、2D-DGE和蛋白质的制备性分离，以供LC MS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.