Wallentine Jeremy C, Kim Ki Kwon, Seiler Charles E, Vaughn Cecily P, Crockett David K, Tripp Sheryl R, Elenitoba-Johnson Kojo S J, Lim Megan S
Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA.
Lab Invest. 2007 Nov;87(11):1113-24. doi: 10.1038/labinvest.3700672. Epub 2007 Sep 17.
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.
基于质谱的蛋白质组学结合液相色谱和生物信息学分析，为蛋白质鉴定提供了一种高灵敏度和高通量的方法。霍奇金淋巴瘤是一种恶性淋巴瘤，其特征是里德-斯腾伯格细胞增殖以及背景反应性淋巴细胞。对里德-斯腾伯格细胞表达和释放的蛋白质进行全面分析，将有助于发现潜在的生物标志物，并增进我们对其发病机制的理解。对L428和KMH2霍奇金淋巴瘤来源的细胞系的三个细胞区室的亚细胞蛋白质组进行了分级分离，并通过反相液相色谱与电喷雾电离串联质谱进行分析。此外，从无血清培养基中提取并分析了霍奇金淋巴瘤来源的L428细胞释放的蛋白质。使用TurboSEQUEST针对UniProt蛋白质数据库（5.26.05；188712个条目）分析肽谱。通过蛋白质印迹分析、免疫荧光显微镜检查和免疫组织化学对鉴定出的一部分蛋白质进行了验证。总共鉴定出1945种蛋白质，其中785种来自细胞质部分，305种来自膜部分，441种来自核部分，414种为释放蛋白，每种蛋白质至少有两个肽段鉴定，错误率<5.0%。来自不同功能组的蛋白质的鉴定反映了里德-斯腾伯格蛋白质组的功能复杂性。鉴定出了在其他癌症中具有先前报道的致癌功能以及涉及霍奇金淋巴瘤的信号通路中的蛋白质。通过蛋白质印迹分析（B-RAF、Erb-B3）、免疫荧光显微镜检查（Axin1、腱生蛋白-X、粘蛋白-2）以及使用组织芯片的免疫组织化学（BRAF、PIM1）对先前未在霍奇金淋巴瘤中证明有表达的选定蛋白质进行了验证。这项研究代表了霍奇金淋巴瘤里德-斯腾伯格细胞表达的蛋白质的首次全面清单，并证明了结合细胞分级分离、蛋白质沉淀、串联质谱和生物信息学分析来全面鉴定可能代表该疾病潜在生物标志物的蛋白质的实用性。
