TCP4: DETECTING AND MAPPING PROTEIN MODIFICATIONS BY MALDI

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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