Mass Spectrometry-Based Glycoproteomic Profiling Identifies Distinct Protein Biomarkers of Hodgkin Lymphoma.
Noah A Brown, Carla M McNeil, Venkatesha Basrur, Fermin Damian, Kevin P Conlon, Delphine S Rolland, Anke van den Berg, Sibrand Poppema, Kojo SJ Elenitoba-Johnson, Megan S Lim. University of Michigan, Ann Arbor; University of Groningen, Netherlands
Background: Hodgkin lymphoma is one of the most frequent lymphomas with 95% representing Classical Hodgkin Lymphoma (CHL) and 5% Nodular Lymphocyte Predominant Hodgkin Lymphoma (NLPHL). While the tumor cells of both entities derive from germinal center B cells, these two lymphomas are clinically and genetically distinct. Overlapping histologic and immunophenotypic features of these lymphomas make this diagnostic distinction challenging in some cases. Since a majority of N-linked glycoproteins are either localized on the membrane or secreted, these proteins are attractive targets for the identification of biomarkers that could aid in the distinction of these two lymphomas.
Design: Using an innovative proteomic strategy targeting N-linked glycosylated proteins, we characterized the glycoproteomic profiles of CHL and NLPHL. Cell lines derived from NLPHL (DEV) and CHL (L428, L1236 and KMH2) were used for solid-phase extraction of N-linked glycopeptides which were identified and quantified using liquid chromatography and tandem mass spectrometry. A subset of proteins was selected for validation using Western blot analyses or flow cytometry.
Results: Glycopeptides from over 250 proteins were identified from each of the four cell lines using false discovery rate of < 4.5 %. This included over 90 cluster designated (CD) proteins. Over 90% of the peptides contained the NXST motif which identifies distinct sites of asparagine glycosylation. Importantly, mass spectrometry data correlated strongly with known proteins expressed by CHL (CD30 and CD15) and NLPHL (CD45, CD19, CD20, CD22, CD79A, CD79B, and IgH). In addition, many novel glycoproteins (adhesion molecules, cytokine receptors and immune regulatory molecules) that were selectively expressed by CHL and NLPHL were identified. Orthogonal analyses by Western blotting and/or flow cytometry corroborated the results obtained by MS/MS with expression of three markers selectively identified in NLPHL and one in CHL.
Conclusions: We identified glycoproteins that had not been previously associated with either CHL or NLPHL and may serve as diagnostic markers. Orthogonal approaches corroborated the results obtained by mass spectrometry. This study demonstrates that mass spectrometry-based glycoproteomic profiling can be used for discovery of novel biomarkers and phenotypic profiling without antibodies.
Category: Special Category - Pan-genomic/Pan-proteomic approaches to Cancer
Monday, February 28, 2011 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 244, Monday Morning