Kaji H, Tsuji T, Mawuenyega K G, Wakamiya A, Taoka M, Isobe T
Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Japan.
Electrophoresis. 2000 May;21(9):1755-65. doi: 10.1002/(SICI)1522-2683(20000501)21:9<1755::AID-ELPS1755>3.0.CO;2-S.
The nematode Caenorhabditis elegans (C. elegans) is the first animal whose whole 97 Mb genome sequence, encoding ca. 19000 open reading frames (ORF's), has been essentially determined. We tried to establish a 2-DE map of the nematode proteome by means of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). A soluble protein fraction of mixed stages of the worm, wild-type strain N2, was applied to 2-D PAGE. After Coomassie Brilliant Blue (CBB) staining, 1200 spots were detected and 140 major spots were excised from the gel and subjected to in-gel digestion with Achromobacter protease I (lysyl endopeptidase). Resulting peptides were analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) followed by peptide mass fingerprinting for protein identification. With this approach we have obtained a two-dimensional electrophoresis (2-DE) protein map in which 69 spots were localized as landmarks for comparison of expression profiles to elucidate the basis of various biological events.
线虫秀丽隐杆线虫（C. elegans）是首个全基因组序列（97 Mb，编码约19000个开放阅读框（ORF））基本确定的动物。我们试图通过二维聚丙烯酰胺凝胶电泳（2-D PAGE）建立线虫蛋白质组的二维图谱。将野生型N2菌株线虫混合发育阶段的可溶性蛋白组分用于二维聚丙烯酰胺凝胶电泳。考马斯亮蓝（CBB）染色后，检测到1200个斑点，从凝胶中切下140个主要斑点，用无色杆菌蛋白酶I（赖氨酰内肽酶）进行胶内消化。所得肽段通过基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS）分析，随后进行肽质量指纹图谱分析以鉴定蛋白质。通过这种方法，我们获得了一张二维电泳（2-DE）蛋白质图谱，其中69个斑点被定位为地标，用于比较表达谱以阐明各种生物学事件的基础。
