小细胞肺癌及其配对的正常肺组织差异表达蛋白质的双相电泳-飞行时间质谱研究

Analysis of differential expression proteins of small cell lung cancer and matched normal lung tissues by 2-DE and MALDI-TOF-MS

目的应用比较蛋白质组学方法分析小细胞肺癌(SCLC)与其配对的正常肺组织差异表达蛋白质,为阐明SCLC发病机制、筛选其早期诊断标志物提供有益的思路。方法应用双相电泳(2-DE)分离6例SCLC及其配对的正常肺组织可溶性总蛋白;应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)获得差异蛋白点的肽质量指纹图谱(PMF);通过Mascot软件查询NCBI或SWISS-PROT数据库鉴定蛋白质。结果每张图谱约检测到800多个蛋白质点。经匹配分析,肺癌组织之间和正常肺组织之间凝胶图像的匹配率分别为75.5%和78.2%;选择14个背景清晰、重复性及分辨率较好、蛋白表达差异明显的点进行质谱分析,结合表观分子量及等电点(pI),初步鉴定了11种(六类)蛋白质:①与蛋白质降解通路有关的蛋白质:蛋白酶体α亚单位3型、蛋白酶体β亚单位2型及β亚单位5型;②自由基/抗氧化剂类:锰-超氧化物歧化酶(Mn-SOD)、过氧化氢酶(CAT)和黄素还原酶(FR);③细胞骨架类:原肌球蛋白-3(Tpm-3);④与能量代谢有关的蛋白质:硫氧还蛋白过氧化物酶(TPX1);⑤分子伴侣:抑制素(PHB),内质网蛋白ER29(Erp29);⑥其他:可溶性NSF黏附蛋白(alpha SNAP)。结论应用2-DE及MALDI-TOF-MS方法分离并初步鉴定了11种(六类)蛋白质;这些蛋白质与SCLC发生发展密切相关,部分可能成为SCLC诊断及治疗的分子靶点。

Objective To investigate the pathogenesis of small cell lung cancer （SCLC） and screen biomarkers for early diagnosis of this disease by analyzing differentially expressed proteins between SCLCs and matched normal lung tissues using comparative proteomic methods. Methods Six sequential patients suffering from SCLC were included in this study. The totally soluble proteins were separated by two dimensional gel electrophoresis（2-DE）and differentially expressed proteins in the protein profiling were analyzed and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS）. Proteins were identified by consulting NCBI or SWISS-PORT database with Mascot software. Results Each gel obtaining about 800 protein spots were seen. The spots in the normal tissues and tumor tissues were matched with an average matching rate of 78.2% and 75.5%, respectively. Fourteen most prominent protein spots were selected for further MALDI-TOF-MS analysis, thirteen protein spots yielded peptide mass fingerprintings （PMFs）, and eleven candidate proteins were identified and further classified into 6 categories according to their functions： （1） proteins associated with ubiquitin-proteasome pathway （UPP） ： proteasome subunit alpha type 3, proteasome subunit beta type 2 and proteasome subunit beta type 5; （2） free radical metabolism/anti-oxidant： Mn-superoxide dismutase （Mn-SOD）, catalase （CAT） and flavin reductase （FR） ; （3） cytoskeleton： tropomyosin-3 （Tpm-3） ; （4） protein associated with energy metabolism： thioredoxin peroxidase 1 （TPX1）;（5） molecular chaperones： prohibitin （PHB） and endoplasmic reticulum protein ER29 （Erp29）;（6） others： alpha soluble NSF attachment protein （alpha-SNAP）. Compared with those of the normal tissues, expression levels of proteins including Mn-SOD, CAT, PHP, ERp29, TXP and FR were upregulated, whereas expression levels of proteins including Tpm-3 and alpha-SNAP were down-regulated in the tumor tissues. Oonclusion Eleven proteins were identified by comparative proteome analysis. Most of them are associated with genesis and development of SCLC and some may serve as potential biomarkers for early detection and targets to SCLC therapy.