Rasmussen H H, van Damme J, Puype M, Gesser B, Celis J E, Vandekerckhove J
Institute of Medical Biochemistry, Aarhus University, Denmark.
Electrophoresis. 1992 Dec;13(12):960-9. doi: 10.1002/elps.11501301199.
Microsequencing of proteins recovered from two-dimensional (2-D) gels is being used systematically to identify proteins in the master human keratinocyte 2-D gel database. To date, about 250 protein spots recorded in human 2-D gel databases have been microsequenced and, of these, 145 are recorded in the keratinocyte database under the entry partial amino acid sequence. Coomassie Brilliant Blue-stained protein spots cut from several (up to 40) dry gels were concentrated by elution-concentration gel electrophoresis, electroblotted onto PVDF membranes and digested in situ with trypsin. Eluting peptides were separated by reversed-phase HPLC, collected individually and sequenced. Computer search using the FASTA and TFASTA programs from Genetics Computer Group indicated that 110 of the microsequenced polypeptides shared significant similarity with proteins contained in the PIR, Mipsx or GenEMBL databases. Only 35 polypeptides corresponded to hitherto unknown proteins. Peptide sequences of all 145 proteins are listed together with their coordinates (apparent molecular weight and pI) in the keratinocyte database.
从二维（2-D）凝胶中回收的蛋白质的微量测序正被系统地用于鉴定人类角质形成细胞主二维凝胶数据库中的蛋白质。迄今为止，人类二维凝胶数据库中记录的约250个蛋白质斑点已进行微量测序，其中145个在角质形成细胞数据库中以部分氨基酸序列条目记录。从几块（最多40块）干凝胶上切下的考马斯亮蓝染色蛋白质斑点通过洗脱浓缩凝胶电泳进行浓缩，电印迹到PVDF膜上，并用胰蛋白酶原位消化。洗脱的肽通过反相HPLC分离，单独收集并测序。使用遗传计算机组的FASTA和TFASTA程序进行的计算机搜索表明，110个微量测序的多肽与PIR、Mipsx或GenEMBL数据库中包含的蛋白质具有显著相似性。只有35个多肽对应于迄今未知的蛋白质。所有145种蛋白质的肽序列及其在角质形成细胞数据库中的坐标（表观分子量和pI）一起列出。
