TCP4: DETECTING AND MAPPING PROTEIN MODIFICATIONS BY MALDI

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。 技术核心项目4 该小组开发了用于绘制蛋白质修饰图谱的灵敏的MALDI方法，对蛋白质乙酰化和泛素化特别感兴趣。第二个项目与公共卫生学院的费尔南多·皮内达合作，涉及开发一种具有细胞分选器和质谱仪功能的超灵敏仪器-目标是能够对单个细胞进行蛋白质图谱分析。这种技术的效用被认为是深远的，而不是描绘进程。 我们的具体目标是： 具体目标1：发展化学衍生化和其他综合方法，以改进多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。用MALDI-MS和MS/MS测定赖氨酸修饰蛋白水解肽的分析和衍生化方法的发展已成功地用于鉴定几种重要的细胞蛋白质的乙酰化位点。在这项建议中，将开发包括胍化和磺化在内的优化的多种衍生化方法，用于蛋白质乙酰化的综合测定。利用化学衍生化技术，将开发一种高效和特异的新策略，用于泛素化位点多肽的浓缩和氨基酸序列测定。 具体目标2：调整和改进目前在内部开发的飞行时间质量分析仪，用于鉴定用于分析蛋白质和蛋白质消化样品的生物气雾剂。MAMS实验室目前参与了一个由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雾剂形式呈现的武器化制剂。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，它将与荷兰TNO Prins Maurits实验室开发的气溶胶收集技术对接。该提案中项目#4的具体目标是利用这种空间聚焦技术来分析所有生物样本作为气溶胶，从中多肽、蛋白质和其他生物分子将被MALDI直接从其位置的单个气溶胶液滴电离 当它们与两个正交的可见光激光相交时被探测到。“空间聚焦”仪器的计算机建模将在Pineda实验室进行。 具体目标3：将生物气雾仪的能力扩展到气雾化单细胞的分析。从单个气溶胶颗粒中电离生物分子的能力提供了进行“分类”过程的机会。虽然气溶胶大小的差异可以直接从定位激光获得，但荧光信号(使用准分子激光激发)已被用于在生物制剂分析中将“生物”气溶胶颗粒与背景区分开来，并可在此用于对含有细胞的气溶胶进行重点分析。事实上，应该可以调整气雾化介质中的细胞浓度，以确保每个液滴有一个细胞。此外，通过将每个质量分析瞬变与其在一定波长范围内的相应荧光光谱一起存储，还应该可以分组和 在混合物中集成来自相似单元的瞬变。这类排序的数据分析算法 还将在Pineda实验室进行分析

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