摘要
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。
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 Peptldent 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.
出处
《癌症》
SCIE
CAS
CSCD
北大核心
2001年第6期575-582,共8页
Chinese Journal of Cancer
基金
中国博士后科学研究基金(中博基[2000] 23号)
湖南省科委研究基金(2000年)
国家自然科学基金(编号:300000
关键词
双向电泳
基质辅助激光解析电离飞行时间质谱
肺肿瘤
鳞癌细胞
NCI-G520
Immobilized pH gradient-two dimensional gel electrophoresis
MALDI-TOF mass spectrometry
Human lung squamous carcinoma cell
Proteome