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High‐temperature LC‐MS/MS of permethylated glycans derived from glycoproteins

Various glycomic analysis methods have been developed due to the essential roles of glycans in biological processes as well as the potential application of glycomics in biomarker discovery in many diseases. Permethylation is currently considered to be one of the most common derivatization methods in MS‐based glycomic analysis. Permethylation not only improves ionization efficiency and stability of sialylated glycans in positive mode but also allows for enhanced separation performance on reversed‐phase liquid chromatography (RPLC). Recently, RPLC‐MS analysis of permethylated glycans exhibited excellent performance in sensitivity and reproducibility and became a widely‐applied comprehensive strategy in glycomics. However, separating permethylated glycans by RPLC always suffers from peak broadening for high‐molecular‐weight branched glycans, which probably due to the low exchange rate between the stationary phase and mobile phase limited by intermolecular interactions of the methyl groups associated with the branching of the glycan structures. In this study, we employed high separation temperature conditions for RPLC of permethylated glycans, thus achieving enhanced peak capacity, improving peak shape, and enhancing separation efficiency. Additionally, partial isomeric separation were observed in RPLC of permethylated glycans at high‐temperature. Mathematical processing of the correlation between retention time and molecular weight also revealed the advantage of high‐temperature LC method for both manual and automatic glycan identification.