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Abstract
Protein post translational modifications (PTMs) refer to the modification by adding a specific moiety to the amino acid side chain after their biosynthesis. Owing to the advances of high-sensitivity mass spectrometry, more than 400 different types of PTMs have been identified, including phosphorylation, ubiquitylation, glycosylation, methylation and acetylation. Biological investigations of the functionalities of different PTMs have revealed their participation in different crucial cellular processes. Dysregulation of PTMs or corresponding regulatory enzymes could result in dysfunction of critical biological processes and lead to occurrence and progression of various diseases. Despite many efforts to disclose protein modifications in cells by quantitative proteomics, the substrate specificity for individual PTM or its regulatory enzyme is still less investigated, which limits our understanding of how the PTM contributes to a specific cellular process. Therefore, there is an urgent need to profile and identify the protein substrates for each single PTM. Here, we reported our work in the study of Histone acetyltransferase 1 (HAT1) substrates, lysine methacrylylation substrates and cysteine (S)-2-carboxypropylation substrates. First, we created a series of engineered HAT1 and found one could efficiently catalyze the acylation and label its protein substrates combined with a clickable acyl-CoA bioorthogonal reporter, 3-azidopropanoyl CoA (3AZ-CoA). We identified hundreds of novel protein substrates of HAT1 by chemoproteomic profiling. Second, we developed a sensitive and robust chemical probe which could selectively label and identify protein methacrylylation substrates. Proteomic identification revealed hundreds of sodium methacrylate and valine dependent methacrylylated proteins as well as HAT1 dependent methacrylylated proteins. Last, we designed and synthesized a bioorthogonal chemical probe which was used to discover a new protein modification, (S)-2- carboxypropylation. Through chemoproteomic profiling, we successfully identified hundreds of protein substrates as well as 120 modification sites. Together, the current work expands the researchers’ toolbox to study protein PTMs, provides valuable insights in understanding the mechanisms of how PTMs regulate cellular processes and offers new directions for the development of therapeutical methods for PTMs related diseases.