Table 2.

Comparison of experimental methods for the verification of irreversible covalent binding.

Assay	Principle	Feature	Readout
Incubation time-dependent activity	Time-dependent increase in inhibition indicates irreversible covalent binding	Easy to operate and low cost	Enzyme inhibition
Washout experiments	The continuous growth of covalent inhibitors is attributed to irreversible covalent binding	Can be used to verify irreversible MOR	Enzyme inhibition
X-Ray crystallography and in situ labeling	Can analyze the covalent binding situation with proteins	Provide more detailed binding information	Ligand‒targets analysis
Chemoproteomic approaches	Can identify specific targeted binding and eliminate non-specific covalent interaction.	Provide more detailed binding information	Ligand‒targets analysis
Alarm NMR	Monitor the DTT-dependent 13C chemical shift changes of the human La antigen	Provide more detailed binding information	Ligand‒targets analysis