Protocol for automated N-glycan sequencing using mass spectrometry and computer-assisted intelligent fragmentation

Biological functions of glycans are intimately linked to fine details in branches and linkages, which make structural identification extremely challenging. Here, we present a protocol for automated N-glycan sequencing using multi-stage mass spectrometry (MSⁿ). We describe steps for release/purification and derivation of glycans and procedures for MSⁿ scanning. We then detail "glycan intelligent precursor selection" to computationally guide MSⁿ experiments. The protocol can be used for both discrete individual glycans and isomeric glycan mixtures. For complete details on the use and execution of this protocol, please refer to Sun et al.,¹ Huang et al.,² and Huang et al.³.