Glycomic analysis of glycans released from glycoproteins using chemical immobilization and mass spectrometry.
Abstact
Protein glycosylation is one of most common protein modifications and is involved in many biological activities. N-linked and O-linked glycosylation not only represent abundant glycan modifications, but also are structurally diverse. Mass spectrometry has emerged as a major method for glycomic analysis. However, glycan extraction from proteins and glycan modification are two critical steps in glycomic analysis of glycans using mass spectrometry. In this protocol, we describe a novel and high-throughput method for isolation and modification of glycans from glycoproteins using a chemoenzymatic approach on solid-phase. Proteins are first immobilized to a solid support and unconjugated molecules are washed away; glycans, while still linked to glycoproteins on the solid support, can be treated enzymatically or chemically on solid phase for glycan derivatization. Glycans are then released from the solid support for analysis by mass spectrometry. The procedures outlined are robust and useful for high-throughput glycomic analysis from complex biological or clinical samples.