Optimization of Ionic Matrix Deposition to Increase the Performance of MALDI‐Based Spatial N‐Glycomics

Ionic matrices for matrix-assisted laser desorption/ionization (MALDI) have demonstrated superior performance compared to traditional matrices for profiling and screening of carbohydrate composition. However, their application in MALDI-MS imaging of N-glycans in tissues remains elusive, in part due to suboptimal matrix application conditions. In this study, we optimized 2,5-dihydroxybenzoic acid/N, N-dimethylaniline (DHB/DMA) ionic matrix spray conditions to enhance the sensitivity of spotted N-glycan standards; however, these conditions were not suitable for analyzing endogenous N-glycans in tissue. To address this, we refined the composition of the ionic matrix by replacing DHB with α-cyano-4-hydroxycinnamic acid (CHCA) and DMA with its hydrochloride salt (DMA·HCl), resulting in superior performance compared to the CHCA matrix, which is the most widely used for spatial N-glycomics. This method enhanced the sensitivity for detecting diverse N-glycan types, including low-abundance sialic acid glycans, while maintaining their endogenous location-a key limitation of liquid-state DMA.