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: 7724688
• 负责人: ROBERT James COTTER
• 金额: $ 37.52万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7724688
• 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

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。 技术核心项目4 这个小组已经开发出了敏感的MALDI方法来绘制蛋白质修饰，特别是在蛋白质乙酰化和泛素化。与公共卫生学院的Fernando Pineda合作的第二个项目涉及开发一种具有细胞分选仪和质谱仪的超灵敏仪器-目标是能够对单个细胞进行蛋白质分析。这种技术的效用被认为是深远的，从剖析的进展。 我们的具体目标是： 具体目标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裂解，从而阐明赖氨酸修饰，特别是乙酰化和泛素化。使用MALDI MS和蛋白水解肽的MS/MS分析以及衍生化方法的发展确定赖氨酸修饰已成功地用于鉴定几种重要细胞蛋白的乙酰化位点。本研究将优化胍基化和磺化等多种衍生化方法，用于蛋白质乙酰化的综合测定。利用化学衍生化技术，将发展一种高效、特异的新策略，用于含泛素化位点的多肽的富集和氨基酸序列测定。 具体目标二：调整和修改目前正在内部开发的飞行时间质量分析仪，用于识别生物气溶胶，以分析蛋白质和蛋白质消化样品。MAMS实验室目前正在参与一个由美国国防高级研究计划局（DARPA）资助的项目，该项目旨在开发一种质谱仪，用于检测和识别以生物气溶胶形式出现的武器化制剂。我们实验室正在为DARPA项目开发的质量分析系统是一种高度“空间聚焦”的仪器，将与荷兰TNO Prins Maurits实验室开发的气溶胶收集技术相连接。本提案中项目#4的具体目标是利用这种空间聚焦”技术来分析作为气溶胶的所有生物样品，其中肽、蛋白质和其他生物分子将通过MALDI直接从单个气溶胶液滴电离， 当它们与两个正交的可见激光相交时被检测到。“空间聚焦”仪器的计算机建模将在皮内达实验室进行。 具体目标3：将生物气溶胶仪器的能力扩展到分析气溶胶化的单细胞。 从单个气溶胶颗粒中分离生物分子的能力提供了进行“分选”过程的机会。虽然气溶胶尺寸的差异可以直接从定位激光器获得，但荧光特征（使用受激准分子激光器激发）已用于在生物制剂分析中将“生物”气溶胶颗粒与背景区分开，并且在此可以用于集中分析含有细胞的气溶胶。事实上，应该可以调节雾化介质中的细胞浓度，以确保每个液滴有一个细胞。此外，通过存储每个质量分析瞬态沿着其在波长范围内的相应荧光光谱，还应该可以对每个质量分析瞬态进行分组和分析。 对来自混合物内类似细胞的瞬态进行积分。这种类型的排序的数据分析算法 分析也将在Pineda labora中进行，