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目的:筛选高转移和低转移肺腺癌细胞中差异表达的膜蛋白,以寻找肺癌的生物学标志物或治疗的潜在靶点。方法:提取具有相同来源、不同转移潜能的肺腺癌SPC-A-1和SPC-A-1sci细胞的膜蛋白,应用核素标记相对和绝对定量(isobaric tags for relative and absolute quantitation,iTRAQ)技术标记膜蛋白,联合纳升液相色谱和串联质谱(nanoliquid chromatography-tandem mass spectrometry,Nano-LC-MS/MS)分离、分析肽段,采用Proteinpilot 4.0软件对蛋白进行鉴定和定量分析,对获得的差异蛋白进行GO(Gene Ontology)分析,应用实时荧光定量-PCR和蛋白质印迹法进行验证。结果:样品进行4次NanoLC-MS/MS,鉴定出符合假阳性率<1%的分别有1413、1374、1297和1351个蛋白,标记率>95%,其中4次都上调的蛋白有27个(膜蛋白20个)、下调的蛋白有32个(膜蛋白25个)。GO分析显示,差异蛋白的分子功能主要集中在细胞骨架蛋白结合、同源蛋白结合和酶结合。生物学进程主要集中在代谢进程和细胞定位。SPC-A-1sci细胞中ITGA3(integrin alpha-3)、MYH9(myosin,heavy chain9)、PLEC1(plectin 1)、HADHA(3-hydroxyacyl-CoA dehydrogenase)、HK1(hexokinase-1)、KTN1(kinectin 1)、ESYT1(extended synaptotagmin-like protein 1)、ALDH18A1(aldehyde dehydrogenase 18 family,member A1)、ATP5A1(ATP synthase alpha-subunit)、LMNB2(lamin-B2)、CAV1(caveolin-1)和CK-19(keratin,typeI cytoskeletal19)mRNA的表达水平以及CLTC(clathrin,heavy chain)、HK1、LMNB2和CK-19蛋白的表达水平均高于SPC-A-1细胞,与质谱定量结果一致。结论:iTRAQ联合NanoLC-MS/MS技术能高通量地筛选与转移相关的膜蛋白,为肺腺癌患者转移的诊断和预后提供可能的生物学标志物或治疗靶点。
OBJECTIVE: To screen differentially expressed membrane proteins in highly metastatic and metastatic lung adenocarcinoma cells in search of a potential biomarker or therapeutic target for lung cancer. Methods: Membrane proteins of lung adenocarcinoma SPC-A-1 and SPC-A-1sci cells with the same source and different metastatic potentials were extracted and purified by using isobaric tags for relative and absolute quantitation (iTRAQ) The proteins were separated and identified by nanoliptic chromatography and tandem mass spectrometry (Nano-LC-MS / MS). The protein was identified and quantified using Proteinpilot 4.0 software. The differential proteins were analyzed by GO (Gene Ontology) and verified by real-time fluorescence quantitative-PCR and Western blotting. Results: Four times of NanoLC-MS / MS showed that 1413, 1374, 1297 and 1351 proteins with the positive rate of <1% and the labeling rate of> 95% were identified. Among them, 27 (20 membrane proteins) and 32 down-regulated proteins (25 membrane proteins). GO analysis showed that the molecular functions of the differentially expressed proteins mainly focused on cytoskeletal protein binding, homologous protein binding and enzyme binding. The biological processes are mainly focused on metabolic processes and cellular localization. ITGA3, MYH9 (myosin, heavy chain9), PLEC1 (plectin 1), HADHA (3-hydroxyacyl-CoA dehydrogenase), HK1 (hexokinase- 1), and KTN1 (kinectin 1) in SPC-A- ), ESYT1 (extended synaptotagmin-like protein 1), ALDH18A1 (member A1), ATP5A1 (ATP synthase alpha-subunit), LMNB2 (lamin-B2), CAV1 (caveolin-1) and CK- keratin, typeI cytoskeletal19) mRNA expression and CLTC (clathrin, heavy chain), HK1, LMNB2 and CK-19 protein expression levels were higher than the SPC-A-1 cells, consistent with the quantitative results of mass spectrometry. Conclusion: iTRAQ combined with NanoLC-MS / MS can screen membrane proteins related to metastasis with high throughput and provide a potential biomarker or therapeutic target for the diagnosis and prognosis of lung adenocarcinoma.