Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL) with a poor prognosis. DLBCL is also clinically heterogeneous, as demonstrated by genomic, transcriptional, translational and post translational molecular characterization. Although several studies have revealed disparate molecular profiles in DLBCL on gene expression, protein abundance, glycosylation occupancy levels, a large scale site-specific N-glycoproteome of DLBCL is still missing, which is crucial to determine N-glycan changes in DLBCL subtypes and explore the functional roles of glycosylation biosynthesis enzymes in DLBCL development.

In this study, we perform proteomic and site-specific N-glycoproteomic quantitative analyses of 20 DLBCL cell lines (7 activate-B-cell-like (ABC) and 13 germinal center B-cell-like (GCB) subtypes). Our results identified and quantified 7800 glycopeptides bearing 457 glycans on 1074 glycosites from 636 proteins. Subsequent analysis based on the glycopeptide abundances classified the DLBCL cell lines into three major subtypes. Integrated proteomic and glycoproteomic analysis identified N-glycosylation changes, which correlated with subtype-specific expression of glycan biosynthesis enzymes. Furthermore, we proposed 3 glyco-processing models regulated by different glycan biosynthesis pathways in DLBCL. Specifically, in model 1 glycopeptides bearing larger glycan with more branches are highly enriched, in model 2 highly sialyated glycopeptides are the most abundant, in model 3 glycopeptides whose glycans own incomplete poly-N-acetyllactosamine (over/under-expressed galactose) show higher expression.

Our results provided a valuable resource and shed light on understanding glycosylation regulations in DLBCL cells and identifying glycan biomarkers for DLBCL diagnosis.