Bioconjugation with Aminoalkylhydrazine for Efficient Mass Spectrometry-Based Detection of Small Carbonyl Compounds

Bioconjugation through oxime or hydrazone formation is a versatile strategy for covalent labeling of biomolecules in vitro and in vivo. In this work, a mass spectrometry-based method was developed for the bioconjugation of small carbonyl compounds (CCs) with an aminoalkylhydrazine to form stable hydrazone conjugates that are readily detectable with electrospray ionization mass spectrometry (ESI-MS). Out of all hydrazine reagents tested, 2-(dimethylamino)­ethylhydrazine (DMAEH) was selected for further analysis due to the fastest reaction rates observed. A thorough study of the reaction kinetics between structurally varied short-chain CCs and DMAEH was performed with the second-order reaction rate constants spanning in the range of 0.23–208 M^–1 s^–1. In general, small aldehydes reacted faster than the corresponding ketones. Moreover, a successful reaction monitoring of a deoxyribose-5-phosphate aldolase-catalyzed reversible retro–aldol cleavage of deoxyribose was demonstrated. Thus, the developed method shows potential also for ESI-MS-based enzyme kinetics studies.