Native top-down mass spectrometry for monitoring the rapid chymotrypsin catalyzed hydrolysis reaction

Enzymes play crucial roles in life sciences, pharmaceuticals and industries as biological catalysts that speed up biochemical reactions in living organisms. New catalytic reactions are continuously developed by enzymatic engineering to meet industrial needs, which thereby drives the development of analytical approaches for realtime reaction monitoring to reveal catalytic processes. Here, taking the hydrolase- chymotrypsin as a model system, we proposed a convenient method for monitoring catalytic processes through native top-down mass spectrometry (native TDMS). The chymotrypsin sample heterogeneity was first explored. By altering sample introduction modes and pHs, covalent and noncovalent enzymatic complexes, substrates and products can be monitored during the catalysis and further confirmed by tandem MS. Our results demonstrated that native TDMS based catalysis monitoring has distinctive strength on real-time inspection and continuous observation, making it a promising tool for characterizing more biocatalysts.

Automated summary

Enzymes play a crucial role as biological catalysts in accelerating biochemical reactions in living organisms. This study proposes a convenient method for monitoring enzymatic catalytic processes using native top-down mass spectrometry. By exploring the heterogeneity of the chymotrypsin sample and using tandem mass spectrometry, the researchers were able to monitor covalent and noncovalent enzymatic complexes, substrates, and products during catalysis. The results demonstrate that this method has the potential to be a promising tool for characterizing biocatalysts.