Spatial Profiling of Glycosaminoglycans (GAGomics) from Laser Microdissected Mouse Brain

• Developed a high-resolution hydrophilic interaction liquid chromatography-coupled cyclic ion mobility-mass spectrometry (HILIC-cIM-MS) method for native heparan sulfate (HS) and chondroitin sulfate (CS) disaccharide analysis. • Achieved enhanced separation of isomeric and epimeric HS and CS disaccharides, as well as lyase-resistant 3- O -sulfated HS tetrasaccharides, using multi-pass cIM. • Enhanced characterization of HS and CS by integrating laser microdissection (LMD)-based tissue collection into the HILIC-cIM-MS workflow. • Demonstrated robust identification and quantification of HS and CS disaccharides from small-scale, spatially resolved mouse brain tissue sections. Developed a high-resolution hydrophilic interaction liquid chromatography-coupled cyclic ion mobility-mass spectrometry (HILIC-cIM-MS) method for native heparan sulfate (HS) and chondroitin sulfate (CS) disaccharide analysis. Achieved enhanced separation of isomeric and epimeric HS and CS disaccharides, as well as lyase-resistant 3- O -sulfated HS tetrasaccharides, using multi-pass cIM. Enhanced characterization of HS and CS by integrating laser microdissection (LMD)-based tissue collection into the HILIC-cIM-MS workflow. Demonstrated robust identification and quantification of HS and CS disaccharides from small-scale, spatially resolved mouse brain tissue sections. 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 (UA) 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.