High-Throughput Analyses of Glycans, Glycosites, and Intact Glycopeptides Using C4-and C18/MAX-Tips and Liquid Handling System.

Protein glycosylation is one of the most common and diverse modifications. Aberrant protein glycosylation has been reported to associate with various diseases. High-throughput and comprehensive characterization of glycoproteins is crucial for structural and functional studies of altered glycosylation in biological, physiological, and pathological processes. In this protocol, we detail a workflow for comprehensive analyses of intact glycopeptides (IGPs), glycosylation sites, and glycans from N-linked glycoproteins. By utilizing liquid handling systems, our workflow could enrich IGPs in a high-throughput manner while reducing sample processing time and human error involved in traditional proteomics sample processing techniques. Together, our workflow enables a high-throughput enrichment of glycans, glycosites, and intact glycopeptides from complex biological or clinical samples. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Enzymatic digestion of glycoproteins using C4-tips Basic Protocol 2: Intact glycopeptide analysis using C18/MAX-tips Basic Protocol 3: Glycan and glycosite analysis.

Chen SY, Clark DJ, Zhang H

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Curr Protoc, 2021, 1 (7)

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