TCP4: DETECTING AND MAPPING PROTEIN MODIFICATIONS BY MALDI

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。该小组开发了敏感的MALDI方法来绘制蛋白质修饰，对蛋白质乙酰化和泛素化特别感兴趣。第二个项目是与公共卫生学院的Fernando Pineda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谱分析。这种技术的实用性被认为是深远的，从分析进展。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，以提高多肽的检测和质谱碎片化，从而阐明赖氨酸修饰，特别是乙酰化和泛素化。利用MALDI质谱和质谱/质谱分析测定赖氨酸修饰以及衍生化方法的发展已成功地用于鉴定几种重要细胞蛋白的乙酰化位点。本课题将优化包括胍化和磺化在内的多种衍生化方法，用于蛋白质乙酰化的综合测定。利用化学衍生化技术，将为含泛素化位点肽的富集和氨基酸测序提供一种高效、特异的新策略。具体目标2：调整和修改目前正在开发的飞行时间质量分析仪，用于鉴定生物气溶胶，用于分析蛋白质和蛋白质消化样品。MAMS实验室目前参与了一项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，用于探测和识别作为生物气溶胶呈现的武器化剂。我们实验室为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结合。该提案中项目#4的具体目标是利用“空间聚焦”技术来分析所有生物样品作为气溶胶，其中肽，蛋白质和其他生物分子将被MALDI电离，直接来自单个气溶胶液滴，其位置在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进行“分类”过程提供了机会。虽然气溶胶大小的差异可以直接从定位激光中获得，但荧光特征（使用准分子激光进行激发）已被用于区分生物剂分析中的“生物”气溶胶颗粒和背景，并且可以在这里用于集中分析含有细胞的气溶胶。事实上，应该有可能调整雾化介质中的细胞浓度，以确保每个液滴有一个细胞。此外，通过在一定波长范围内存储每一个质量分析瞬态及其各自的荧光光谱，还可以将混合物中类似细胞的瞬态进行分组和整合。这种分类分析的数据分析算法也将在皮内达实验室进行

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