Comprehensive identification of proteins in Hodgkin lymphoma-derived Reed–Sternberg cells by LC-MS/MS

Abstract

Mass spectrometry-based proteomics in conjunction with liquid chromatography and bioinformatics analysis provides a highly sensitive and high-throughput approach for the identification of proteins. Hodgkin lymphoma is a form of malignant lymphoma characterized by the proliferation of Reed–Sternberg cells and background reactive lymphocytes. Comprehensive analysis of proteins expressed and released by Reed–Sternberg cells would assist in the discovery of potential biomarkers and improve our understanding of its pathogenesis. The subcellular proteome of the three cellular compartments from L428 and KMH2 Hodgkin lymphoma-derived cell lines were fractionated, and analyzed by reverse-phase liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Additionally, proteins released by Hodgkin lymphoma-derived L428 cells were extracted from serum-free culture media and analyzed. Peptide spectra were analyzed using TurboSEQUEST® against the UniProt protein database (5.26.05; 188 712 entries). A subset of the identified proteins was validated by Western blot analysis, immunofluorescence microscopy and immunohistochemistry. A total of 1945 proteins were identified with 785 from the cytosolic fraction, 305 from the membrane fraction, 441 from the nuclear fraction and 414 released proteins using a minimum of two peptide identifications per protein and an error rate of <5.0%. Identification of proteins from diverse functional groups reflected the functional complexity of the Reed–Sternberg proteome. Proteins with previously reported oncogenic function in other cancers and from signaling pathways implicated in Hodgkin lymphoma were identified. Selected proteins without previously demonstrated expression in Hodgkin lymphoma were validated by Western blot analysis (B-RAF, Erb-B3), immunofluorescence microscopy (Axin1, Tenascin-X, Mucin-2) and immunohistochemistry using a tissue microarray (BRAF, PIM1). This study represents the first comprehensive inventory of proteins expressed by Reed–Sternberg cells of Hodgkin lymphoma and demonstrates the utility of combining cellular subfractionation, protein precipitation, tandem mass spectrometry and bioinformatics analysis for comprehensive identification of proteins that may represent potential biomarkers of the disease.