# Spatial Profiling of Glycosaminoglycans (GAGomics) From Laser Microdissected Mouse Brain

## TL;DR

The paper introduces a new method to study complex sugars in mouse brain tissue with high precision and spatial resolution.

## Contribution

A novel workflow combining LMD, HILIC, and cIM-MS for spatially resolved analysis of HS and CS disaccharides in small tissue samples.

## Key findings

- The workflow enables separation and identification of isomeric HS and CS disaccharides.
- Rare HS disaccharides with saturated uronic acid and 3-O-sulfated tetrasaccharides were detected.
- Quantitative analysis of disaccharide abundances was achieved in microdissected tissue sections.

## Abstract

Glycosaminoglycans (GAGs) are linear, negatively charged polysaccharides composed of repeating disaccharide units. Heparan sulfate (HS) and chondroitin sulfate (CS) are highly sulfated GAG classes, ubiquitously expressed in mammalian tissues, that play critical roles in cellular signaling, tissue homeostasis, and disease progression. Aside from their biological importance, the structural analysis of HS and CS remains limited to bulk tissue analysis due to their extensive heterogeneity, structural complexity, and the presence of isomers and epimers. In this work, we developed an integrated workflow combining laser microdissection (LMD), hydrophilic interaction liquid chromatography (HILIC), and cyclic ion mobility mass spectrometry (cIM-MS) for the identification and quantification of HS and CS disaccharides from small-scale and spatially resolved mouse brain tissue sections. Through sequential enzymatic digestion of HS and CS chains from the same sample, we profiled not only the common disaccharides that serve as structural signatures for HS and CS, but also rarely detected HS disaccharides containing saturated uronic acid residues, as well as lyase-resistant 3-O-sulfated HS tetrasaccharides. HILIC enabled the separation of HS and CS disaccharides based on their composition and hydrophilicity, while cIM-MS further enhanced the resolution of positional isomers. Quantitative analysis using linear calibration curves revealed disaccharide abundances in small-scale tissue sections collected by LMD. Overall, our finding highlighted the merit of the LMD-HILIC-cIM-MS workflow for HS and CS analysis in spatial GAGomics and its potential for biomarker discovery and therapeutic application studies.

•Developed a high-resolution HILIC-cIM-MS method for native HS and CS analysis.•Achieved enhanced separation of HS and CS isomeric pairs.•Integrated an LMD-based tissue collection method into the HILIC-cIM-MS workflow.•Demonstrated spatial identification and quantification methods of HS and CS.

Developed a high-resolution HILIC-cIM-MS method for native HS and CS analysis.

Achieved enhanced separation of HS and CS isomeric pairs.

Integrated an LMD-based tissue collection method into the HILIC-cIM-MS workflow.

Demonstrated spatial identification and quantification methods of HS and CS.

We present a streamlined laser microdissection (LMD)-hydrophilic interaction liquid chromatography (HILIC)-cyclic ion mobility-mass spectrometry (cIM-MS) workflow for high-resolution, native analysis of heparan sulfate (HS) and chondroitin sulfate (CS) from spatially resolved, small-scale mouse brain tissue, enabling improved identification and quantification of HS and CS.