小细胞肺癌细胞系NCI-H446蛋白质表达谱的建立

Establishment of Protein Profile of Human Small Cell Lung Cancer Cell Line NCI-H446

背景与目的:小细胞肺癌(smallcelllungcancer,SCLC)是一种侵袭性极强的恶性肿瘤,具有增长迅速、早期转移等特点。目前公开的数据库中尚未见到小细胞肺癌的双向电泳参考图谱及其蛋白表达谱。本研究目的是建立高分辨率的小细胞肺癌细胞系NCI-H446细胞双向凝胶电泳图谱,并初步分析其蛋白质表达情况。方法:用固相pH梯度双向凝胶电泳技术(IPG-DALT)分离NCI-H446细胞总蛋白,凝胶银染显色,ImageMaster2D图像分析系统分析,从凝胶中选取分离较好的蛋白质点,应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术和数据库搜索鉴定蛋白质。结果:获得了背景清晰、分辨率和重复性较好的双向凝胶电泳图谱,三块胶平均蛋白质点数为1506±74,匹配点数为1412±56,匹配率达93.4%,三块胶蛋白质点在位置上有较好的重复性,不同胶间蛋白质点在IEF方向偏差是(0.96±0.27)mm,在SDS-PAGE方向偏差是(1.24±0.41)mm。胶内酶解-肽质指纹图分析鉴定了58个蛋白质,其中有原癌蛋白、细胞周期调控和信号转导相关蛋白质。结论:建立了小细胞肺癌细胞系NCI-H446双向电泳参考图谱,并应用质谱技术鉴定了部分蛋白质点,为进一步构建其蛋白质表达数据库提供了基础。

BACKGROUND &OBJECTIVE: Small cell lung cancer (SCLC) is particular ly aggressive, and characterized by rapid growth and early metastasis. At presen t, there is no data concerning SCLC two-dimensional polyacrylamide gel electrop horesis (2-DE) reference map,and its protein profiles in public databases. This study was to establish a well-resolved, reproducible 2-DE map of proteome in SCLC cell line NCI-H446, and analyze its protein profiles. METHODS: Two-DE was applied to separate the total proteins of NCI-H446 cells, which were then silv er-stained in the gel. Well-seperated protein spots were selected from the gel by ImageMaster 2D analysis system. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), peptide map fingerprinting ( PMF),and database searching were used to identify the protein spots. RESULTS: Cl ear,well-resolved, reproducible 2-DE patterns of proteome in NCI-H446 cells w ere obtained. The average protein spots of 3 gels were 1 506±74; and 1 412±56 spots were matched with an average matching rate of 93.4%. The average deviatio n of spot position was (0.96±0.27) mm in IEF direction, and (1.24±0.41) mm in SDS-PAGE direction indicating relatively good reproducibility of the protein sp ots. Fifty-eight proteins were identified, certain proteins were products of on cogenes, and others were involved in cell cycle regulation, and signal transduct ion. CONCLUSIONS: A reference map of NCI-H446 cells was established,certain pro teins were identified by MALDI-TOF-MS and PMF. These data will be useful for e stablishing human SCLC proteome database.