Abstract
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.
Highlights
Various bioconjugation reactions have become an essential route to investigate the chemical and biological molecules in vitro and in vivo.[1]
These reactions are older than many other bioconjugation reactions and have already been studied by German scientists in the late 1800s.3−6 In particular, hydrazines are commonly used as nucleophiles due to their high reactivity toward carbonyl compounds (CCs), forming stable hydrazone conjugates
The results indicate that the developed electrospray ionization mass spectrometry (ESI-MS) method is suitable for the simultaneous detection and further quantitation of substrates and products of enzyme-catalyzed reactions and holds a vast potential for ESI-MS-based enzyme kinetics assays, especially in the case of small molecules that are difficult to detect with conventional atmospheric-pressure ionization (API) techniques
Summary
Various bioconjugation reactions have become an essential route to investigate the chemical and biological molecules in vitro and in vivo.[1]. A mass spectrometry based method was developed for the bioconjugation of small carbonyl compounds (CCs) with an aminoalkylhydrazine to form stable hydrazone conjugates, which are readily detectable with electrospray ionization mass spectrometry (ESI-MS). To compare the efficiency of different compounds promoting hydrazone/oxime-based bioconjugation of various CCs at neutral pH, we selected four hydrazines (DNPH, MPH, HBA, and DMAEH) for initial reaction kinetics studies (Figure 2). Kool et al previously studied the reaction rates of aliphatic vs aromatic CCs and confirmed that simple aliphatic compounds react more rapidly than aromatic ones, and that the steric effects are moderate to small, except in case of very bulky aldehydes.[27,28] It was concluded that electron-deficient CCs generally react faster than electron-rich ones in hydrazone formation This is in line with our observations. The results indicate that the developed ESI-MS method is suitable for the simultaneous detection and further quantitation of substrates and products of enzyme-catalyzed reactions (here demonstrated for the DERA enzyme) and holds a vast potential for ESI-MS-based enzyme kinetics assays, especially in the case of small molecules that are difficult to detect with conventional API techniques
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