Numerous biological events need to be orchestrated at an epigenetic level upon defining cellular fate. Among the key epigenetic regulators are protein methyltransferases (PMTs), which methylate specific Arg/Lys residues with S-adenosyl-L- methionine (SAM) as a cofactor. Whereas many recombinant PMTs are active in vitro, their cellular activities can largely depend on the presence of distinct PMT isoforms or complexes. Such context- dependent complexity makes it challenging to solely rely on conventional methods to elucidate the targets of PMTs. The flexible substrate-recognizing patterns of PMTs, together with their highly- conserved SAM-binding motifs, also made it challenging to develop PMT inhibitors with potency and selectivity. A long-term goal of the project is to elucidate and manipulate methyltransferase- involved epigenetic for disease diagnosis and treatments. The objective of this application is to use SAM mimics as probes to profile cell-type-specific substrates of PMTs and to perturb PMTs in a specific manner. Here an integrative Bioorthogonal Profiling of Protein Methylation (BPPM) will be implemented to dissect the substrates of designated PMTs in relevant biological contexts. A subtype of methylation events associated with specific biological processes will be further validated with well-established methods. Their functional roles will be examined in the context of cancer malignancy or stem cell reprogramming. In parallel, stable SAM analogues will be pursued as target-specific inhibitors against PMTs. The completion of this proposal will unambiguously reveal the substrates of multiple PMTs and characterize high-quality PMT inhibitors in relevant cellular contexts. The impact of these substrate-profiling and inhibitor-identifying strategies further lie n their general applicability to other methyltransferases.