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。 科布雷蛋白质组学核心实验室有几个质谱仪，可以帮助科布雷研究人员和他们的学生。有两台Thermo-Finnigan LTQ线性四极离子陷阱液相色谱质谱计(LC MS)，一台Thermo-Finnigan LTQ Deca XP离子陷阱LC MS和一台能够高精度质量测量的Thermo-Finnigan LTQ Orbitrap LC MS。所有仪器都有电喷雾电离(ESI)作为主要入口，LTQ仪器在纳米喷雾电离中运行，使用熔融二氧化硅制成的微型LC柱，该柱也用作纳米喷雾尖端。纳米旋光技术将多肽分析的检测下限提高到飞摩尔范围。核心继续与科布雷调查人员个人进行讨论，以说明核心的能力以及如何利用核心来解决科布雷调查人员的科学问题。布莱恩·巴里夫博士(生物学助理教授)在他的博士后研究期间接受了广泛的蛋白质组学培训，他也参与了CORE协助几名Cobre研究人员的工作。巴里夫博士的实验室专门为大规模分析蛋白质磷酸化位点而设计。他的实验室经常使用的方法正在被纳入科布雷调查员的样本分析。核心中心有工作人员，也有标准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，但许多工作需要在设计实验时进行，以获得最佳的质谱学数据。马修斯和巴里夫博士的大部分工作都是在实验设计和数据分析中与研究人员互动，以识别蛋白质和定位翻译后修饰。 蛋白质组学的质谱学分析 下列调查员提交了核心在2010年4月1日至2010年3月31日期间测量和完成的样本： 调查员委任部 派：贾森·博滕，助理教授，医学-免疫生物学分部。 +Joe Klaus研究生 +菲尔·艾森豪尔技师 +Joanne Russo技术人员 Pi：拉尔夫·巴德教授医学免疫生物学 迪夫。 +Andreas Koenig博士后研究员 +谢丽尔·柯林斯技术人员 派：克里斯托弗·休斯顿助攻。医学感染性教授 疾病 +Adam Sateriale研究生 派：玛丽安娜·马特拉伊特助攻。微生物学&教授 分子遗传学 Pi：Cory Teuscher医学免疫生物学教授 迪夫。 派：加里·沃德教授微生物学与 分子遗传学 +梁成龙研究生 核心的一个重要部分是与研究生和博士后实习生合作，让他们接触蛋白质组学，并让他们参与样本测量和数据分析组件。 玛丽安娜·马特拉伊特和加里·沃德的项目重点是弓形虫。Matrajt博士已经准备并提交了Work Performance的一篇手稿，另一篇来自Ward博士的手稿于今年发表在《公共科学图书馆·病原体》杂志上。克里斯·休斯顿的这个项目希望继续他在来UVM之前就开始的有针对性的全球蛋白质组实验。拉尔夫·巴德和安德烈亚斯·凯尼格同样也在寻求使用蛋白质组学方法寻找与半胱氨酸酶和非典型T细胞受体结合的蛋白质伙伴。Jason Botten的项目专注于识别与ArenaVirus蛋白相关的宿主蛋白，最近获得了NIH R21奖，该奖项主要涉及这些蛋白质组分析。Cory Teuscher已经开始识别与组胺受体相互作用的蛋白质，最近获得了NIH R01奖，这些蛋白质组学实验是其中的一个重要目标。 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)。

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 several mass spectrometers available to assist COBRE investigators and their students. There are two Thermo-Finnigan LTQ linear quadrupole ion trap liquid chromatograph - mass spectrometers (LCMS) a Thermo-Finnigan LCQ Deca XP ion trap LCMS, and a Thermo-Finnigan LTQ Orbitrap LCMS capable of high accuracy mass measurement. All instruments have electrospray ionization (ESI) as the primary inlet, and the LTQ instruments are operated in nanospray ESI with micro-LC columns made of fused silica that also function as the nanospray tip. Nanospray improves detection limits for peptide analysis into the femtomole range. The Core continued discussion with the individual COBRE investigators to relate the capabilities of the Core and how the Core could be used to address scientific problems of COBRE investigators. Bryan Balliff, Ph.D. (Assistant Professor of Biology) who has extensive training in proteomics during his postdoctoral studies has also become involved in the Core in assisting several COBRE investigators. Dr. Ballif's laboratory is particularly designed for large scale analysis of protein phosphorylation sites. The methods that his laboratory uses routinely are being incorporated into COBRE investigator sample analyses. Personnel are available in the Core and standard protocols are in place to perform digests of proteins to peptides for proteomic analysis by LCMS, but much of the effort needs to occur in designing experiments to obtain the best mass spectrometry data. Much of the effort by Drs. Matthews and Ballif has been interacting with investigators in experiment design and data analysis for protein identification and location of posttranslational modifications. Proteomics Analyses by Mass Spectrometry The following investigators submitted samples that have been measured and completed by the Core during the period 4/1/2009  3/31/2010: Investigator Appointment Department PI: Jason Botten Assistant Professor Med-Immunobiology Div. +Joe Klaus Graduate Student +Phil Eisenhauer Technician +Joanne Russo Technician PI: Ralph Budd Professor MedImmunobiology Div. +Andreas Koenig postdoctoral fellow +Cheryl Collins Technician PI: Christopher Huston Assist. Prof Medicine  Infectious Disease +Adam Sateriale Graduate Student PI: Mariana Matrajt Assist. Prof Microbiology & Molecular Genetics PI: Cory Teuscher Professor MedImmunobiology Div. PI: Gary Ward Professor Microbiology & Molecular Genetics +Jackie Leung Graduate student An important part of the Core is working with graduate student and postdoctoral trainees, exposing them to proteomics and involving them in the sample measurement and data analysis components. The projects by Mariana Matrajt and Gary Ward focus on Toxoplasma gondii. One manuscript has been prepared and submitted by Dr. Matrajt from work performed, another from Dr. Ward was published this year in PLOS Pathogens. The project by Chris Huston is looking to continue targeted global proteomic experiments he began prior to coming to UVM. Ralph Budd and Andreas Koenig similarly are seeking to find protein binding partners to caspases and atypical T-cell receptors using proteomics approaches. Jason Botten's projects focus on the identification of host proteins that associate with arenavirus proteins and was recently awarded an NIH R21 award that heavily involved these proteomic analyses. Cory Teuscher has begun work to identify proteins that interact with histamine receptors and was recently awarded an NIH R01 award in which these proteomics experiments were an important aim. 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).