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. Two projects are being carried out at the Resource, to provide useful data and to serve as a training platform for Ms. Seo. (1) Characterization Of Vimentin fragments and their post-translational modifications under external stimuli and (2) Characterization Of Fas Associating Factor 1 (FAF 1) and posttranslational modifications in response to heat shock. Methods developed at the BUSM MS Resource for sample preparation and analysis and data interpretation are being applied, with the focus being on MALDI and ESI QoTOF MS and MS/MS and Fourier Transform Ion Cyclotron Mass Spectrometry (FT-ICR-MS). Life phenomena, such as cellular development, proliferation, aging, apoptosis, carcinogenesis, and disease, are regulated by protein differential expression, post-translational modification (PTM) of proteins, protein-protein interactions and so on. PTMs of a protein can determine its activity state, localization, turnover and interactions with other proteins. Despite the importance of PTMs, the study on PTMs has many limitations such as sensitivity, resolution, sequence coverage and the need for high throughput analysis. Especially, we need better resolution for differentiating some kinds of PTMs. Post-translational modifications of Vimentin fragments in HUVECs were globally defined by MALDI-TOF MS and nano-LC-ESI-Q-TOF MS (in Korea). However, these instruments currently do not have the ability to confirm PTMs due to their low accuracy (-50 ppm). Even when immunoblot analysis was employed, difficulties were encountered in confirmation of PTMs, in the cases of some of PTMs such as acetylation, trimethylation, phosphorylation, and sulfonation. At BUSM, the QoTOF MS experiments have been repeated with higher performance instruments, using well-developed methods for the mass spectral analysis and data interpretation. Any still ambiguous details of the characterization of PTMs are being addressed using a home-built 7T FT-ICR-mass spectrometer. Analysis of proteins using FT-ICR-mass spectrometry 1. Characterization of PTMs using MS/MS analysis of post-translational modifications such as phosphorylation, acetylation, oxidation, methylation and sulfonation 2. Methodology development for enrichment of low abundant modified proteins 3. Methodology development for almost 100% peptide sequence coverage of protein for comprehensive understanding. The exact location and diversity of PTMs over the whole peptide sequences will provide some useful information to evaluate the biological effects in the cellular system. This proteomic technique is needed and applied to the biotechnology related industry, pharmaceutical industry and understanding of life phenomena. This research can be used to find out the biomarkers of disease, identify the proteins regulating diseases, diagnose the disease and develop new drugs.
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