CAJ | 학술논문

目的：建立和优化肿瘤蛋白质组研究的方法系统，并分析人肺鳞癌细胞蛋白质组。方法：用固相 pH梯度双向凝胶电泳分离人肺鳞癌细胞系 NCI H520总蛋白，银染显色， PDQuest 2DE软件分析，对部分蛋白质点用基质辅助激光解析电离飞行时间质谱 ( matrix assisted laser desorption/ionization time of flying mass spectrometry， MALDI TOF MS) 测定其胶内酶解后的肽质指纹图谱，用 PeptIdent软件查询 SWISS PROT数据库。结果：获得了分辨率和重复性均较好的双向电泳银染图谱，图像分析探测到 3块胶的平均蛋白质点数为 (1146± 116)，平均匹配的点数为 (851± 95)，匹配率达 73.7％， 3块胶在蛋白质点位置上具有较好的重复性， IEF方向的平均偏差为 (1.52± 0.22)mm， SDS PAGE方向的平均偏差为 (1.97± 0.13) mm。随机取 60个蛋白质点进行胶内原位酶解 - 质谱指纹图分析得到了 54个蛋白质点的肽质指纹图，查询数据库初步鉴定了 44个蛋白质，其中部分是与细胞周期有关的蛋白，部分是与信号传导有关的蛋白，部分是与癌基因相关的蛋白。结论：通过该研究建立了一套分辨率和重复性均较好的固向 pH梯度双向凝胶电泳分离细胞总蛋白和银染胶内原位酶解后 MALDI TOF MS肽质指纹图分析鉴定方法。通过该方法系统分析人肺鳞癌细胞 NCI H520蛋白质组成分而获得的资料将有益于人肺鳞癌细胞蛋白质组数据库的建立，从而为肺鳞癌的诊断、预防和治疗提供依据。
목적：건립화우화종류단백질조연구적방법계통，병분석인폐린암세포단백질조。방법：용고상 pH제도쌍향응효전영분리인폐린암세포계 NCI H520총단백，은염현색， PDQuest 2DE연건분석，대부분단백질점용기질보조격광해석전리비행시간질보 ( matrix assisted laser desorption/ionization time of flying mass spectrometry， MALDI TOF MS) 측정기효내매해후적태질지문도보，용 PeptIdent연건사순 SWISS PROT수거고。결과：획득료분변솔화중복성균교호적쌍향전영은염도보，도상분석탐측도 3괴효적평균단백질점수위 (1146± 116)，평균필배적점수위 (851± 95)，필배솔체 73.7％， 3괴효재단백질점위치상구유교호적중복성， IEF방향적평균편차위 (1.52± 0.22)mm， SDS PAGE방향적평균편차위 (1.97± 0.13) mm。수궤취 60개단백질점진행효내원위매해 - 질보지문도분석득도료 54개단백질점적태질지문도，사순수거고초보감정료 44개단백질，기중부분시여세포주기유관적단백，부분시여신호전도유관적단백，부분시여암기인상관적단백。결론：통과해연구건립료일투분변솔화중복성균교호적고향 pH제도쌍향응효전영분리세포총단백화은염효내원위매해후 MALDI TOF MS태질지문도분석감정방법。통과해방법계통분석인폐린암세포 NCI H520단백질조성분이획득적자료장유익우인폐린암세포단백질조수거고적건립，종이위폐린암적진단、예방화치료제공의거。
Objective：This study was designed to establish and optimize the research methods for proteome,and to analyze the proteome components of human lung squamous carcinoma cell line NCI H520. Methods： A series of methods, including immobilized pH gradient two dimensional polyacrylamide gel electrophoresis(2DE), silver staining, PDQuest 2DE analysis software, peptide mass fingerprint based on matrix assisted laser desorption/ionization time of flying mass spectrometry (MALDI TOF MS) and SWISS PROT database searching, were used to separate and indentify the proteome of human lung squarmous carcinoma cell line NCI H520. Results： The good 2DE pattern including resolution and reproducibility was obtained. After silver staining, the 2DE image analysis by PDQuest 2DE software had detected average of 1146± 116 spots, and 851± 95 spots were matched. The average matching rate was 73.3％ . There had a good reproducibility of spot position in 2DE map, with average deviation in IEF direction of 1.52± 0.22 mm， while in SDS PAGE direction it was 1.97± 0.13 mm. Sixty spots were incised from silver staining gel randomly and digested in gel by TPCKtrypsin. Fifty four peptide mass fingerprints (PMF) maps were obtained by MALDI TOF MS. The typic peptide masses were searched in the SWISS PROT database by PeptIdent software. Forty four proteins were preliminarily identified. Some of them were cell cycle related proteins such as Cyclin H, some were signal transduction related proteins such as mitogen activated protein kinase, protein kinase C and receptor protein tyrosine kinase ERBB 2, some were oncogene related proteins such as Ras related protein RAB 36, etc. Conclusions： The main proteome research system including IPG 2DE, image analysis, MALDI TOF MS derived PMFs and database searching has been established. The data of NCI H520 obtained by above methods will be useful for the establishment of human lung squamous cell proteome database.