TCP4: DETECTING AND MAPPING PROTEIN MODIFICATIONS BY MALDI

TCP4：通过 MALDI 检测和绘制蛋白质修饰

• 批准号: 7622842
• 负责人: ROBERT James COTTER
• 金额: $ 35.19万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7622842
• 关键词: Acetylation; Aerosols; Algorithms; Amino Acid Sequence; Biological; Cells; Chemicals; Collaborations; Collection; Computer Retrieval of Information on Scientific Projects Database; Computer Simulation; Data Analyses; Detection; Development; Ensure; Fluorescence; Funding; Goals; Grant; Housing; Individual; Institution; Laboratories; Lasers; Lysine; Maps; Methods; Modification; Netherlands; Peptides; Positioning Attribute; Post-Translational Protein Processing; Process; Protein Acetylation; Protein Analysis; Proteins; Public Health Schools; Range; Research; Research Personnel; Research Project Grants; Resources; Sampling; Site; Sorting - Cell Movement; Source; Space Models; Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Systems Analysis; Techniques; Technology; Thinking; Time; Ubiquitination; United States National Institutes of Health; aerosolized; improved; instrument; interest; mass analyzer; mass spectrometer; novel strategies; particle; size

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. Technology Core Projects 4 This group has developed sensitive MALDI methods for mapping protein modifications, with a special interest in protein acetylation and ubiquitination. A second project, in collaboration with Fernando Pineda of the School of Public Health, involves developing an ultrasensitive instrument with aspects of a cell sorter and mass spectrometer - the goal is to be able to perform protein profiling of individual cells. The utility of such a technique is thought to be far reaching, from profiling the progression. Our specific aims are: Specific Aim 1: Develop chemical derivatization and other comprehensive methods for improving the detection and mass spectral fragmentation of peptides leading to the elucidation of lysine modifications, particularly acetylation and ubiquitylation. Determination of lysine modifications using MALDI MS and MS/MS analysis of proteolytic peptides and the development of derivatization methods have been successfully used in the identification of the acetylation sites of several important cellular proteins. In this proposal, optimized multiple derivatization methods including guanidination and sulfonation will be developed for the comprehensive determination of protein acetylation. Using chemical derivatization techniques, a novel strategy with high efficiency and specificity will be developed for the enrichment and amino acid sequencing of ubiquitylation sitecontaining peptides. Specific Aim 2: Adapt and modify a time-of-flight mass analyzer currently being developed in house for the identification of bioaerosols for the analysis of proteins and protein digest samples. The MAMS laboratory is currently involved in a project, funded by the Defense Advanced Research projects Agency (DARPA), for the development of a mass spectrometer for detecting and identifying weaponized agents presented as bioaerosols. The mass analysis system being developed in our laboratory for the DARPA project is a highly "space focusing" instrument that will be interfaced with aerosol collection technology developed at the TNO Prins Maurits Laboratory in the Netherlands. This specific aim for Project #4 in this proposal is to utilize this space focusing" technology to analyze all biological samples as aerosols, from which peptides, proteins and other biological molecules will be ionized by MALDI, directly from individual aerosol droplets whose positions are detected as they intersect two orthogonal visible lasers. Computer modeling for the "space focusing" instrument will be carried out in the Pineda laboratory. Specific Aim 3: extend the capabilities of the bioaerosol instrument to the analysis of aerosolized single cells. The ability to ionize biological molecules from individual aerosol particles provides an opportunity to carry out a "sorting" process. While differences in aerosol size can be obtained directly from the positioning lasers, fluorescent signatures (using an excimer laser for excitation) have been used to distinguish "biological" aerosol particles from background in bioagent analyses and can be utilized here to focus analyses on aerosols containing cells. It should in fact be possible to adjust cell concentrations in the aerosolization medium to ensure that there is one cell per droplet. In addition, by storing each and every mass analysis transient along with its respective fluorescence spectrum over a range of wavelength, it should also be possible to group and integrate transients from similar cells within a mixture. Data analyses algorithms for this type of sorting analyses will also be carried out in the Pineda labora

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此，可以在其他清晰的条目中表示。列出的机构是 对于中心，这不一定是调查员的机构。 技术核心项目4 该组开发了对绘制蛋白质修饰的敏感MALDI方法，对蛋白质乙酰化和泛素化具有特别的兴趣。第二个项目与公共卫生学院的费尔南多·派迪达（Fernando Pineda）合作，涉及开发具有细胞分散器和质谱仪方面的超敏化仪器 - 目标是能够对单个细胞进行蛋白质分析。这种技术的实用性被认为远远不超过进展。 我们的具体目的是： 具体目的1：开发化学衍生化和其他综合方法，以改善肽的检测和质谱片段化，从而阐明赖氨酸的修饰，尤其是乙酰化和泛素化。使用MALDI MS和MS/MS分析蛋白水解肽的测定以及衍生化方法的开发已成功地用于鉴定几种重要细胞蛋白的乙酰化位点。在此提案中，将开发出优化的多种衍生化方法，包括鸟化和硫化，以全面地确定蛋白质乙酰化。使用化学衍生化技术，将开发一种具有高效率和特异性的新型策略，以富集和氨基酸测序的泛素化位置肽。 具体目标2：适应和修改当前正在内部开发的飞行时间分析仪，以鉴定生物素化剂，以分析蛋白质和蛋白质摘要样品。 MAMS实验室目前参与了由国防高级研究项目局（DARPA）资助的项目，用于开发用于检测和识别呈现为生物美感的武器的质谱仪。在我们的DARPA项目实验室中开发的质量分析系统是一种高度“空间焦点”的工具，将与荷兰TNO Prins Maurits实验室开发的气溶胶收集技术相连。该提案中项目＃4的具体目的是利用该空间的焦点“技术来分析所有生物样品作为气溶胶，肽，蛋白质和其他生物分子将直接由MALDI引起，直接从单个气溶胶液滴中引起其位置。 当它们相交两个正交可见激光时被检测到。 PINEDA实验室将进行“焦点”仪器的计算机建模。 具体目标3：将生物溶质仪器的能力扩展到雾化单细胞的分析。 从单个气溶胶颗粒中电离生物分子的能力为执行“分类”过程提供了机会。虽然可以直接从定位激光器获得气溶胶尺寸的差异，但已使用荧光特征（使用准分子激光进行激发）可将“生物学”气溶胶颗粒与生物启动分析的背景区分开来，并可以在此处利用在此处使用包含气溶胶的气体粒子。细胞。实际上，应该可以调整雾化培养基中的细胞浓度，以确保每滴有一个细胞。另外，通过将每个质量分析及其各自的荧光光谱存储在一定范围的波长上，也应该可以分组和分组和 整合来自混合物中相似细胞的瞬变。数据分析此类排序算法 分析也将在Pineda Labara中进行