N-linked glycosylation enrichment for in-depth cell surface proteomics and the classification of diffuse large B-cell lymphoma subtypes

The classification of histologically similar yet molecularly distinct tumors into specific subtypes remains a clinically challenging task. Classification of such tumors into distinct entities based on their cell surface protein expression profiles has been hindered by the lack of an unbiased global approach. Here we use N-glyco FASP, a recently developed mass spectrometric approach based on lectin-enrichment of N-linked glycoproteins, in conjunction with a super-SILAC based quantitative strategy, on patient derived diffuse large B-cell lymphoma cell lines. We mapped 2383 glycosites on more than 1300 proteins, which were highly enriched for cell membrane proteins. The resulting sub-proteome was highly enriched for cell membrane proteins. This N-glyco sub-proteome alone allowed the segregation of the ABC from the GCB subtypes of diffuse large B-cell lymphoma, which before gene expression studies had been considered one disease entity. Encouragingly, many of the glycopeptides driving the segregation belong to proteins previously characterized as segregators in a deep proteome study of these subtypes (S. J. Deeb et al MCP 2012 PMID 22442255). This conforms to the high correlation that we observed between the expression level of the glycosites and their corresponding proteins. Detailed examination of glycosites and glycoprotein expression levels uncovered, amongst other interesting findings, enrichment of transcription factor binding motifs, including known NF-kappa-B related ones. Thus, enrichment of a class of post-translationally modified peptides can classify cancer types as well as reveal cancer specific mechanistic changes.