Profiling of Permethylated Mucin O-glycans Using Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the profiling of mucin glycans that offers high sensitivity and throughput. Here, we provide a detailed protocol for the analysis of O-linked glycans from mucins using MALDI-TOF MS, encompassing glycan release, desalting, permethylation, and data acquisition and analysis. This study shows that desalting based on ion exchange chromatography leads to improved recovery of larger glycans and better quality of the produced spectra compared to solid-phase extraction using porous graphitized carbon (PGC). Additionally, this work suggests that a longer permethylation reaction time than currently employed might be necessary for efficient modification of sialylated structures. We also demonstrate the importance and necessity of fragmentation data and prior knowledge of the O-glycan biosynthetic pathways for the accurate identification of glycan composition and structure and discuss the inherent benefits and limitations that MALDI-TOF presents in the analysis of mucin O-glycans.