人肺腺癌细胞系A549细胞双向电泳-飞行时间质谱研究

A proteomic study on human lung adenocarcinoma cell line A549 by 2-DE and MALDI-TOF-MS

目的初步分析人肺腺癌细胞系A549细胞蛋白质表达情况,从蛋白组水平探讨肺腺癌发病的分子机制。方法应用2-DE技术对人肺腺癌细胞系A549细胞总蛋白进行分离,获得蛋白质表达谱;应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)结合生物信息学进行蛋白质鉴定。结果3块胶平均蛋白斑点数为1 138±49,平均匹配点数为986±32,匹配率为85.8%。随机选取背景清晰、分辨清楚、蛋白含量较高的20个斑点进行MALDI-TOF-MS分析,共获得了18个肽质量指纹图谱(PMF)。将PMFs质量数据通过Aldente软件查询SWISS-PROT数据库,根据匹配片段及氨基酸序列覆盖率等,初步鉴定出15种蛋白质。根据功能,这些蛋白质可分为:①基本代谢相关的酶类:果糖2-磷酸醛缩酶、视网醛脱氢酶1(RALDH1)、吡多醛激酶;②细胞骨架类:蛋白细胞角蛋白8(CK8)、-β2链微管蛋白;③应激相关蛋白:蛋白二硫化物异构酶A3(PDIA3);④信号转导分子:膜联蛋白1(ANX1)、膜联蛋白4(ANX4);⑤分子伴侣:热休克蛋白60(HSP60)、热休克蛋白-β1、抑制素(PHB);⑥转录及翻译相关蛋白:不均一性核糖核蛋白H(hnRNP H);⑦杂类:Rgr protein、NPM、PCBP1。结论应用2-DE/MALDI-MS方法获得了较为理想的人肺腺癌细胞系A549细胞2-DE蛋白质表达谱,初步鉴定了15种蛋白质。

To analyze protein profiles in human lung adenocarcinoma cell line A549 cell and to investigate molecular mechanism of pulmonary adenocarcinoma genesis at the level of proteomics. Methods Two dimensional gel electrophoresis （2-DE） was used to separate totally soluble proteins extracted from A549 cell, and peptide mass fingerprintings （PMFs） were obtained and then some spots on 2-DE gels were identified by matrixassisted laser desorption/ionization time-of-flight mass spectrometry （ MALDI-TOF-MS ） combined with bioinformatics. Results The average total number of proteins spots was 1138±49, average paired spots 986±32, and matched rate 85.8% among the three gels. 20 spots with high resolution and reproducibility were analyzed using MALDI-TOF-MS, 18 spots yielded PMFs, and 15 proteins were preliminarily identified and further classified into 7 categories based on their functions： enzymes related to cell metabolism： fructose-bisphosphate aldolase A, retinal dehydrogenase 1 （RALDH1）, pyridoxal kinase; cytoskeleton proteins ： type II cytoskeletal 8 （CK8）, tubulin beta2 chain;proteins related to stress： protein disulfide-isomerase A3 （PDIA3）; signal tranduction molecules.. annexin AI（ANX1）and annexin A4（ANX2）; chaperones： 60 ku heat shock protein（HSP60）, heat shock protein beta-l, prohibitin （PHB）; proteins related to transcription and translation： heterogeneous nuclear ribonucleoprotein H （hnRNP H）; others： Rgr protein, nucleophosmin （NPM）, poly （ rC）-binding protein 1 （PCBP1）.Conclusion A clear, well-resolved protein profile in A549 cell was obtained by 2-DE and 15 proteins were identified using PMFs.