双向凝胶电泳-飞行时间质谱法分析人肺鳞癌细胞NCI-H520蛋白质组成分

Analysis of Human Lung Squamous Carcinoma Cell Line NCI-H520 Proteome by Two-Dimensional Electrophoresis and MALDI-TOF-Mass Spectrometry

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.