Determination of Site-specific Glycan Heterogeneity on Glycoproteins
Overview
Affiliations
The comprehensive analysis of protein glycosylation is a major requirement for understanding glycoprotein function in biological systems, and is a prerequisite for producing recombinant glycoprotein therapeutics. This protocol describes workflows for the characterization of glycopeptides and their site-specific heterogeneity, showing examples of the analysis of recombinant human erythropoietin (rHuEPO), α1-proteinase inhibitor (A1PI) and immunoglobulin (IgG). Glycoproteins of interest can be proteolytically digested either in solution or in-gel after electrophoretic separation, and the (glyco)peptides are analyzed by capillary/nano-liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). If required, specific glycopeptide enrichment steps, such as hydrophilic interaction liquid chromatography (HILIC), can also be performed. Particular emphasis is placed on data interpretation and the determination of site-specific glycan heterogeneity. The described workflow takes approximately 3-5 d, including sample preparation and data analysis. The data obtained from analyzing released glycans of rHuEPO and IgG, described in the second protocol of this series (10.1038/nprot.2012.063), provide complementary detailed glycan structural information that facilitates characterization of the glycopeptides.
Hu M, Xu K, Yang G, Yan B, Yang Q, Wang L Clin Proteomics. 2025; 22(1):6.
PMID: 39923034 PMC: 11807306. DOI: 10.1186/s12014-025-09525-9.
Xie Y, Butler M Methods Mol Biol. 2024; 2853:249-263.
PMID: 39460925 DOI: 10.1007/978-1-0716-4104-0_16.
Quantitative profiling of N-glycosylation of SARS-CoV-2 spike protein variants.
Xie Y, Butler M Glycobiology. 2023; 33(3):188-202.
PMID: 36723867 PMC: 10114651. DOI: 10.1093/glycob/cwad007.
Chen Y, Yao Q, Zeng X, Hao C, Li X, Zhang L Front Oncol. 2022; 12:1014159.
PMID: 36408150 PMC: 9671074. DOI: 10.3389/fonc.2022.1014159.
Glycosylation and the global virome.
Pegg C, Schulz B, Neely B, Albery G, Carlson C Mol Ecol. 2022; 32(1):37-44.
PMID: 36217579 PMC: 10947461. DOI: 10.1111/mec.16731.