Abstract
Global analysis of lymphoma genome integrity and transcriptomes tremendously advanced our understanding of their biology. Technological advances in mass spectrometry-based proteomics promise to complete the picture by allowing the global quantification of proteins and their post-translational modifications. Here we use N-glyco FASP, a recently developed mass spectrometric approach using lectin-enrichment, in conjunction with a super-SILAC approach to quantify N-linked glycoproteins in lymphoma cells. From patient-derived diffuse large B-cell lymphoma cell lines, we mapped 2383 glycosites on 1321 protein groups, which were highly enriched for cell membrane proteins. This N-glyco subproteome 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, among 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.
Highlights
Correct classification of cancer subtypes is a long-standing aim for any heterogeneous diagnostic category and is a necessary basis for rational treatment
Our previous study had shown that these five activated B-cell-like DLBCL (ABC) and five Germinal-center B-celllike DLBCL (GCB) cell lines can be segregated very clearly by principal component analysis based on their global protein expression profiles (3)
This finding confirms that these cell lines are good representatives of ABC and GCB lymphomas and attractive models to investigate if closely related tumor subtypes can be characterized by a quantitative post-translational modifications (PTM)-based approach
Summary
Cell Culture—DLBCL cell lines (HBL1, OciLy3, RIVA, TMD8, U2932, BJAB, DB, HT, SUDHL-4, SUDHL-6) were grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 20% bovine serum and Penicillin/Streptomycin (1:1000). For N-glycosylation enrichment, tryptic peptides were transferred to a new filtration unit They were mixed with a mixture of lectins (ConA, WGA, and RCA lectins) and incubated for 60 mins. The captured peptides were treated with PNGase F in H218O, which leaves a characteristic mass shift on the previously glycosylated site (18) This was followed by elution and measurement of the deglycosylated peptides (14). Peptides were eluted with a linear gradient of 8 –30% buffer B (80% acetonitrile and 0.5% acetic acid) at a flow rate of 200 nl/min over 145 min. This was followed by 20 min from 30 to 60% buffer B. We analyzed the MaxQuant output data with the Perseus tools, which are available in the MaxQuant environment
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