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Steric-Free Bioorthogonal Profiling of Cellular Acetylation and Glycosylation Via a Fluorine-Selenol Displacement Reaction (FSeDR)
Zhao, Yue
Zhao, Yue
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2024-05
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Chemistry
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http://dx.doi.org/10.34944/dspace/10570
Abstract
The dynamic and low-abundance nature of protein post-translational modifications (PTMs) poses significant challenges for their global detection and identification. Traditional immunoaffinity enrichment and previous bioorthogonal chemistry-based approaches are limited by their inherent constraints. Our research introduces a novel steric-free bioorthogonal strategy utilizing a fluorine-selenol displacement reaction (FSeDR) to overcome these limitations. Building upon a previously reported fluorine-thiol displacement reaction (FTDR), we have innovated selenol as a next-generation fluorine-displacement probe, enabling efficient labeling and imaging of acetylation and glycosylation events at the cellular level.Our methodology involved the incorporation of fluorinated acetyl-CoA analogs onto peptide and protein substrates in solution by key acetyltransferases and cells through pro-metabolite treatment. The incorporation was validated through mass spectrometric analysis and in-gel western blot with FTDR. To optimize the reaction conditions and efficiency, the FSeDR reaction was developed by collaborative graduate students. This bioorthogonal reaction was coupled with SILAC-based quantitative proteomics to profile acetylation substrate proteins globally in a representative prostate cancer cell line, PC3.
Expanding the scope of our research, we delved into protein glycosylation modifications. By incorporating fluorine-substituted monosaccharides into multiple cell lines, we achieved cellular imaging and labeling of protein glycosylation. This innovative approach allowed us to study the glycosylation patterns in various cellular contexts, providing insights into the dynamic nature of glycosylation and its implications in cellular signaling and disease pathology.
The findings reveal a powerful fluorine-based toolbox for probing PTMs, with the potential to systematically study their roles in biological and pathology-related cellular signaling regulation. The FSeDR strategy not only provides a steric-free labeling platform for substrate proteins but also paves the way for future advancements in the functional annotation of PTMs. This research holds significant promise for diagnostic and therapeutic applications, offering a new lens through which to understand the intricate web of cellular signaling.
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Accompanied by one .xlsx file: 1) Zhao_temple_0225E_171/APPENDIX_TABLE_1_Identified_Proteins_in_Proteomics.xlsx
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