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

佛蒙特州免疫生物学/传染病 CTR：核心 C：蛋白质组学分析

• 批准号: 7959815
• 负责人: Dwight E Matthews
• 金额: $ 7.98万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7959815
• 关键词: Address; Appointment; Caspase; Cells; Centers of Research Excellence; Cessation of life; Communicable Diseases; Complex; Computer Retrieval of Information on Scientific Projects Database; Data; Data Analyses; Detection; Disease; Electrospray Ionization; Funding; Grant; Human Resources; Immunobiology; Individual; Institution; Ions; Laboratories; Liquid substance; Location; Manuscripts; Mass Spectrum Analysis; Measurement; Measures; Medicine; Metabolic; Microbiology; Molecular Genetics; Operative Surgical Procedures; Organism; Peptides; Phosphorylation; Phosphorylation Site; Physiological; Post-Translational Protein Processing; Postdoctoral Fellow; Preparation; Protein Binding; Proteins; Proteomics; Protocols documentation; Publications; Research; Research Personnel; Resolution; Resources; Sampling; Science; Signal Transduction; Silicon Dioxide; Site; Source; Students; Techniques; Time; Toxoplasma gondii; United States National Institutes of Health; Vermont; Work; design; graduate student; improved; instrument; mass spectrometer; professor; protein complex; research study; thymocyte; ward

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 newly acquired Thermo-Finnigan LTQ Orbitrap LCMS capable of high resolution 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. 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. The easy analyses are just that, but many of the samples presented come with a complex set of issues that need to be worked through for a successful analysis. Much of the effort is designing how the mass spectrometry experiment should be done to obtain the data that will answer the question posed by the COBRE investigator. 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 both difficult and time consuming. Dr. Matthews and his staff have worked closely with investigators on data analysis. Proteomics Analyses by Mass Spectrometry The following investigators submitted samples that have been measured and completed by the Core during the period 4/1/2008  3/31/2009: Investigator Appointment Department PI: Jonathan Boyson Assistant Professor Surgery +Alan Chant postdoctoral fellow PI: Ralph Budd Professor MedImmunobiology Div. +Andreas Koenig postdoctoral fellow PI: Christopher Huston Assist. Prof Medicine  Infectious Disease PI: Mariana Matrajt Assist. Prof Microbiology & Molecular Genetics +Maria Julia Figueras Graduate student PI: Mercedes Rinc¿n Associate Professor MedImmunobiology Div. +Tina Thornton Research Assoc. PI: Gary Ward Professor Microbiology & Molecular Genetics +Qing Tang Graduate student +Aoife Heaslip 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 and their students focus on Toxoplasma gondii. One manuscript has been prepared and submitted by Dr. Matrajt from work performed. The project by Mercedes Rinc¿n and Tina Thornton focused on cell signaling via protein phosphorylation in thymocytes for cellular control. This work has resulted in a publication in 2008 in Science. Jonathan Boyson is looking to confirm identification of a sub-unit of a protein complex he is currently studying in semi-invariant NKT (iNKT) cells, and Chris Huston is looking to continue targeted global proteomic experiments he began prior to coming to UVM. Ralph Budd and Andreas Koenig similarly are hoping to find protein binding partners to caspases using proteomics approaches. 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).

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有几个质谱仪可用于协助COBRE研究人员和他们的学生。 有两台Thermo-Finnigan LTQ线性四极杆离子阱液相色谱-质谱仪（LCMS），一台是Thermo-Finnigan LCQ Deca XP离子阱LCMS，另一台是新获得的Thermo-Finnigan LTQ Orbitrap LCMS，能够进行高分辨率质量测量。 所有仪器都具有电喷雾电离（ESI）作为主要入口，并且LTQ仪器在具有由熔融二氧化硅制成的微型LC柱的纳米喷雾ESI中操作，所述微型LC柱也用作纳米喷雾尖端。 Nanospray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。 简单的分析就是这样，但是所提供的许多示例都带有一系列复杂的问题，需要通过这些问题进行成功的分析。 大部分工作是设计如何进行质谱实验，以获得回答COBRE调查员提出的问题的数据。 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。 蛋白质组学实验的数据溢出导致死亡，将数据减少到有意义的蛋白质鉴定或特定翻译后修饰的位置既困难又耗时。 马修斯博士和他的工作人员在数据分析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2008年4月1日期间由核心测量和完成的样本  2009年3月31日： 研究者 任命 部 PI：Jonathan Boyson助理教授 手术 +Alan Chant 博士后 PI：Ralph Budd 教授 MedImmunobiology Div. +Andreas Koenig 博士后 PI：克里斯托弗休士顿助攻。教授 医学  传染 疾病 PI：Mariana Matrajt 帮忙教授 微生物学& 分子遗传学 +Maria Julia Figueras 研究生 PI：Mercedes Rinc？n 医学副教授Immunobiology Div. +蒂娜·桑顿 研究助理 PI：加里沃德 教授 微生物学& 分子遗传学 +唐青 研究生 +Aoife Heaslip 研究生 核心的一个重要组成部分是与研究生和博士后学员合作，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。 Mariana Matrajt和加里沃德及其学生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和Tina Thornton的项目专注于通过胸腺细胞中的蛋白质磷酸化进行细胞控制的细胞信号传导。 这项工作已于2008年在《科学》杂志上发表。 Jonathan Boyson正在确认他目前正在半不变NKT（iNKT）细胞中研究的蛋白质复合物的亚单位的鉴定，Chris休士顿正在寻求继续他在来UVM之前开始的有针对性的全球蛋白质组学实验。 Ralph Budd和Andreas Koenig同样希望使用蛋白质组学方法找到半胱天冬酶的蛋白质结合伴侣。 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。