Mass Spectrometric Enzyme Histochemistry Method Developed for Visualizing In Situ Cholinesterase Activity in Mus musculus and Drosophila melanogaster

Abstract

Enzyme histochemistry facilitates enzyme activity visualization in situ; however, as it is a color-based method, molecular quantification is prohibitive. This study aimed to develop a semiquantitative, mass spectrometry imaging (MSI)-based enzyme histochemistry method to determine endogenous cholinesterase (ChE) activity. Using deuterium-labeled acetylcholine (ACh-d9) as a substrate to distinguish ACh-d9 and choline-d9 from endogenous acetylcholine and choline, respectively, the heterogeneous localization of de novo ChE activity was visualized using MSI, devoid of interferences from in situ factors. Furthermore, a tissue inhibitor assay involving two ChE inhibitors in the mouse brain revealed specific ChE inhibition in the corpus callosum. To the best of our knowledge, this study is the first to report a visualization method for total ChE activity in the ganglia and abdomen in Drosophila melanogaster, indicating its applicability among different animals. The present results provide novel insights into the applicability of enzyme histochemistry via MSI to the metabolism of low-molecular-weight organic compounds (i.e., “small molecules”) and semiquantitative capability, suggesting that MSI enzyme histochemistry may become a powerful tool for heterogeneous tissue studies.

Mass Spectrometric Enzyme Histochemistry for Choline Acetyltransferase Reveals De Novo Acetylcholine Synthesis in Rodent Brain and Spinal Cord

“…Applying the normalization formula (1), the ionization efficiency ratio of ACh-d9 and choline-d9 on the spinal cord section was examined and found to be ε = 3, which was the same as that reported for the mouse brain and fly sections in our earlier study (Figure S5). 33 Furthermore, the image visualization of ChAT activity using eq 1 confirmed strong ChAT activity, particularly in the anterior horn and ventral root (Figure 3). Additionally, ChAT activity was also observed in the posterior horn and dorsal root.…”

“…The method directly visualized ChE activity on mouse brain and fly tissues and revealed unique and quantitative ChE activity in detail. 33 In this study, we extend our earlier work and developed a ChAT activity visualization method using matrix-assisted laser desorption/ionization MSI (MALDI-MSI), as ChAT visualization is difficult with conventional enzyme histochemistry based on color reaction. To validate our proof of principle, we developed a method using choline chloride-trimethyl-d9 (choline-d9) and acetyl-CoA as substrates and by adding ChE inhibitor to prevent degradation of products.…”

“…where ε is the ionization efficiency ratio of ACh-d9 to choline-d9, which was estimated to be 3 for the mouse brain in our previous work. 33 The ionization efficiency in spinal cord tissue was also evaluated in this study.…”

“…Mass spectrometry imaging (MSI) has attracted attention as a new tool for enzyme histochemistry in recent years. , MSI can obtain localized images of molecules such as proteins and metabolites by mass spectrometry on the tissue section. − MSI-based enzyme histochemistry is expected to allow visualization of various enzymatic reactions without secondary reactions. However, MSI-based enzyme histochemistry is still under development and has been applied only to hydrolases such as protease, phospholipase, and cholinesterase or to phosphatases and kinases, which have already been investigated using conventional enzyme histochemistry. ,− We recently developed the semiquantitative MSI-based enzyme histochemical method for cholinesterase (ChE) using deuterium-labeled ACh as substrate. The method directly visualized ChE activity on mouse brain and fly tissues and revealed unique and quantitative ChE activity in detail …”

“…However, MSI-based enzyme histochemistry is still under development and has been applied only to hydrolases such as protease, phospholipase, and cholinesterase or to phosphatases and kinases, which have already been investigated using conventional enzyme histochemistry. ,− We recently developed the semiquantitative MSI-based enzyme histochemical method for cholinesterase (ChE) using deuterium-labeled ACh as substrate. The method directly visualized ChE activity on mouse brain and fly tissues and revealed unique and quantitative ChE activity in detail …”