Diamond Deborah L, Zhang Yanni, Gaiger Alexander, Smithgall Molly, Vedvick Thomas S, Carter Darrick
Ciphergen Biosystems Inc., Fremont, California, USA.
J Am Soc Mass Spectrom. 2003 Jul;14(7):760-5. doi: 10.1016/s1044-0305(03)00265-4.
The identification of proteins differentially expressed between cancer and normal cells is vital for the development of cancer diagnostics, therapeutics and vaccines. Using a ProteinChip Biomarker System (Ciphergen Biosystems, Fremont, CA) which combines ProteinChip technology with time-of-flight mass spectrometry, we have developed a simple method to screen and identify differentially secreted proteins from tumor cell lines. Mass spectra of the range of proteins secreted from normal B-cells were generated along with those secreted from Epstein-Barr virus transformed B-cells. A mass peak at m/z = 4972.1 that was highly over-represented in the transformed B-cell line was chosen for identification and purified by reversed phase chromatography with concomitant monitoring of fractions by SELDI-TOF MS. The resulting purified protein was digested with trypsin and the peptide masses derived from the SELDI-TOF spectrum were used to search the public databases for protein identification. Fragment matching of the resulting peptides identified the protein as thymosin beta-4. Using LC-electrospray ionization MS/MS, the identity of this protein was confirmed. Thymosin beta-4 is a known marker in LCLs establishing the utility of this method to discover and identify proteins differentially expressed between cancers and their matched normal counterparts.
鉴定癌症细胞与正常细胞之间差异表达的蛋白质对于癌症诊断、治疗和疫苗的开发至关重要。我们使用一种将蛋白质芯片技术与飞行时间质谱相结合的蛋白质芯片生物标志物系统（Ciphergen生物系统公司，加利福尼亚州弗里蒙特），开发了一种简单的方法来筛选和鉴定肿瘤细胞系中差异分泌的蛋白质。我们生成了正常B细胞分泌的一系列蛋白质的质谱图，以及爱泼斯坦-巴尔病毒转化的B细胞分泌的蛋白质的质谱图。选择在转化的B细胞系中高度富集的m/z = 4972.1处的一个质谱峰进行鉴定，并通过反相色谱法进行纯化，同时用表面增强激光解吸电离飞行时间质谱监测各馏分。将得到的纯化蛋白用胰蛋白酶消化，从表面增强激光解吸电离飞行时间质谱图中获得的肽质量用于搜索公共数据库以鉴定蛋白质。所得肽段的片段匹配鉴定该蛋白为胸腺素β-4。使用液相色谱-电喷雾电离串联质谱，证实了该蛋白的身份。胸腺素β-4是淋巴母细胞系中的一种已知标志物，确立了该方法在发现和鉴定癌症与其匹配的正常对应物之间差异表达的蛋白质方面的实用性。
