The nucleophilic thiol-alkyne addition reaction can form a covalent thiovinyl bond with cysteine reactivity specific,as the classical standards for cysteine-reactive warheads in activity-based probes (ABPs).Utilizing this classical reactivity property, we designed two potentially reactive unnatural amino acids containing alkyne moiety in its side chain, named as 4-propargyloxy-L-phenylalanine (pPR) and enrichable 4-propynyloxy-L-phenylalanine (epPR),for capturing deubiquitinases or ubiquitin-like protein specific proteases in the ubiquitin (Ub) and ubiquitin-like (Ubl) modification.The technology was extended to polyubiquitin chains through Sortase A mediated transpeptidation using reduced N6-((2-azidoacetyl)glycyl)-L-lysine (AzGGK) to triglycine tail (GGK),constructing ubiquitin conjuates,such as di diubiquitin (Diub). Rare codon recoding techonology (RCR) can be used to incorporation unnatural amino acid to mammalian cell, utilizing the rare codons to introduce multiple unnatural amino acids into proteins, creating multireactive and high-efficiency crosslinking probes. Here,we genetically encoded and sortase-mediated transpeptidation to develope crosslinkable and enrichable Ub/Ubl/Diub ABPs in E.coli and HEK293T cells.Meanwhile,the alkyne cysteine specificity of the covalent binding to the deubiquitinases allows for the preliminary screening of pan and irreversible inhibitors.Since most chemical Ub/Ubl/Diub ABPs are generally not cell-permeable, we performed functional complementation studies using genetically encoded biosynthetic Ub/Ubl/Diub ABPs. Meanwhile, the insertion of unnatural amino acids at multiple sites of UFM1 multi-reactive crosslinking probe was realized to be expressed in living cells by using the RCR technology, providing a novel methodology for comprehensive mapping of UFM1 interaction networks.