Mapping Enzyme Activity on Tissue by Functional Mass Spectrometry Imaging

Abstract

Enzymes are central components of most physiological processes, and are consequently implicated in various pathologies. High-resolution maps of enzyme activity within tissues therefore represent powerful tools for elucidating enzymatic functions in health and disease. Here, we present a novel mass spectrometry imaging (MSI) method for assaying the spatial distribution of enzymatic activity directly from tissue. MSI analysis of tissue sections exposed to phospholipid substrates produced high-resolution maps of phospholipase activity and specificity, which could subsequently be compared to histological images of the same section. Functional MSI thus represents a new and generalisable method for imaging biological activity in situ.

Keywords: PLA2; enzymes; functional assays; lipids; mass spectrometry imaging.

Figure 1

Mass‐spectrometry‐based enzyme assays are sensitive and powerful. Activity assay of porcine pancreatic PLA₂ on phosphatidylcholine 34:1 isomers by nESI‐MS. A) The temporal change in abundance of the substrate (PC 16:0/18:1, red) and LPC products. B) Averaged mass spectrum from the shaded area in (A). C) Depletion of PC 18:1/16:0 (red), and formation of LPCs. Averaging over the shaded area yields the mass spectrum shown in (D).

Figure 2

fMSI of N. subfulva venom gland, showing the distribution of PLA₂ activity against two different substrates. A) Optical image of a 7 μm section of venom gland tissue. B) MALDI‐MSI ion map. C) Averaged MALDI mass spectrum in the absence of lipid substrate. Application of PC 16:1/16:1 (D–F) or PC 15:0/18:1‐d₇ ((G)–(I)) with the MALDI matrix enables acquisition of fMSI ion maps of the substrate (blue) and PLA₂ product (yellow) for each section ((E), (H)), along with their average spectra ((F), (I)). Scale bar: 2 mm.

Figure 3

MSI and histology of the N. subfulva venom gland post‐fMSI. A) Section of the N. subfulva venom gland after paraffin removal before substrate and matrix deposition (left); an H&E stain obtained following fMSI and MSI data acquisition and matrix removal (right). B) Averaged mass spectrum from MSI analysis of the same N. subfulva venom gland section post‐fMSI acquisition, showing abundant signals from three‐finger toxins known to be abundant in the venom. C) Spatial distribution of endogenous compounds in the N. subfulva venom gland, including three‐finger toxins with possible cytotoxic and neurotoxic activities. Intensities of extracted ions (±1 Da) from normalized spectra are shown as heat maps across the tissue section. Scale bar: 2 mm.