Table 1.

A summarization of the pros and cons for surrogate substrate assays, natural substrate assays and ABPP assays.

Assay	Pros	Cons
Surrogate substrate assays	Cost-effectiveness; easy detection of the product; enzymatic reaction progress can be monitored in real-time.	Binding affinities of enzymes can be attenuated due to artificial substrate; inhibitor potency (e.g., IC50 values) might be affected by use of different surrogate substrates.
LC–MS-based assays (natural substrate assays)	Highly sensitive and accurate.	Costly; less high throughput; cannot monitor enzymatic reaction progress in real-time; complex experimental procedures (e.g., lipid extraction); limited samples can be acquired and measured.
Fluorometric glycerol assay (natural substrate assays)	Using natural substrate (2-AG); enzyme inhibition can be tested in a more physiological condition; enzymatic reaction progress can be monitored in real-time; application in high throughput screening.	False-positive reduction: compounds interacting with glycerol should be excluded; experimental procedure is less straightforward.
ABPP	Without the need of substrate; activity and selectivity can be measured in one single experiment; both in vitro and in vivo activity/selectivity can be measured; a selectivity profile across entire proteome can be measured.	An effective activity-based probe is required; gel-based ABPP assay is less high throughput.