Spatial glycomics: a multi-enzyme MALDI imaging protocol

Why the glycome needs its own protocol

Many metabolites and lipids can be imaged directly by MALDI. Glycogen and protein-bound N-glycans cannot: they first have to be cut on the tissue with enzymes into fragments the instrument can detect, and every step before acquisition (how fast tissue is frozen, how thick it is cut, how evenly enzymes and matrix go down) shows up in the data.

One digestion, two halves of the glycome

The core of the method is a multi-enzyme (dual-enzyme) digestion, which lets one section cover glycogen and N-glycans together: spatial glycomics rather than a single glycan class. Two enzymes go onto the tissue together, in one solution and one spray: isoamylase, which cuts glycogen into its glucose chains, and PNGase F, which releases N-linked glycans from their proteins. After a single incubation, the same section yields both.

On the glycogen side: where glycogen sits, how much there is (relative abundance), its chain-length distribution, read from chains that differ by one glucose unit (for example DP6, maltohexaose), and information about how those chains branch. On the N-glycan side: individual N-glycan species, each with its own pattern, from hepatocyte-wide to regional foci to portal-tract structures. The paper describes this as an integrated workflow for glycome analysis.

The workflow, step by step

In fresh-frozen mouse liver: (1) rapid harvesting and freezing; (2) cryostat sectioning at the right thickness and position; (3) tissue preparation and the dual-enzyme digestion; (4) CHCA matrix sprayed with an HTX M5 sprayer, then MALDI-MSI in positive-ion mode at 50 µm raster steps; and (5) data processing in flexImaging and SCiLS Lab to place the glycan maps in biological context.

Spots of HRP (a glycoprotein) and rabbit liver glycogen serve as internal standards, so each run carries its own check that both enzymes worked. The protocol is written to transfer to other tissues, species and MALDI platforms.

Why it matters

The method is built to define spatial glycomic heterogeneity tied to liver function and dysfunction (the paper demonstrates it in wild-type mouse liver), and the workflow is built for reproducibility and sensitivity. Because JoVE publishes the procedure on video, another lab can watch each step rather than guess from a methods paragraph.

My role

First author. Spatial glycomics (glycogen and N-glycans) was one of the three assay families I ran routinely at CASBR, alongside spatial metabolomics and lipidomics, across cancer, neurodegeneration and rare metabolic disease projects.