IDENTIFYING PROTEINS FROM 2-DIMENSIONAL GELS BY MOLECULAR MASS SEARCHING OF PEPTIDE-FRAGMENTS IN PROTEIN-SEQUENCE DATABASES
IDENTIFYING PROTEINS FROM 2-DIMENSIONAL GELS BY MOLECULAR MASS SEARCHING OF PEPTIDE-FRAGMENTS IN PROTEIN-SEQUENCE DATABASES
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DOI：
10.1073/pnas.90.11.5011
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发表时间：
1993-06-01
影响因子：
11.1
通讯作者：
WATANABE, C
中科院分区：
文献类型：
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作者：
HENZEL, WJ;BILLECI, TM;WATANABE, C
描述了一种通过二维凝胶电泳分离已知蛋白质的快速鉴定方法，该方法使用肽片段的分子质量来搜索蛋白质序列数据库。这些肽是通过原位还原、烷基化和胰蛋白酶消化从二维凝胶中电印迹的蛋白质而产生的。质量是确定在亚皮摩尔水平的基质辅助激光解吸/电离质谱法的未分级消化。已经开发了一个计算机程序，用于在蛋白质序列数据库中搜索与测量质量相匹配的单个蛋白质的多个肽。为了确保包含最新的数据库更新，每次执行程序时都会生成整个数据库的理论摘要。该方法便于同时处理大量二维凝胶点。该方法应用于大肠杆菌粗提取物的二维凝胶，该凝胶被电印迹到聚偏二氟乙烯膜上。随机选择10个点进行分析。只有三个肽团，每个蛋白质都是从91,000多个蛋白质序列中唯一鉴定出来的。所有鉴定均通过第二次印迹中相同斑点的同步n端测序进行验证。其中一个点含有一个n端阻断蛋白，需要酶裂解、肽分离和Edman降解来确认其身份。
A rapid method for the identification of known proteins separated by two-dimensional gel electrophoresis is described in which molecular masses of peptide fragments are used to search a protein sequence database. The peptides are generated by in situ reduction, alkylation, and tryptic digestion of proteins electroblotted from two-dimensional gels. Masses are determined at the subpicomole level by matrix-assisted laser desorption/ionization mass spectrometry of the unfractionated digest. A computer program has been developed that searches the protein sequence database for multiple peptides of individual proteins that match the measured masses. To ensure that the most recent database updates are included, a theoretical digest of the entire database is generated each time the program is executed. This method facilitates simultaneous processing of a large number of two-dimensional gel spots. The method was applied to a two-dimensional gel of a crude Escherichia coli extract that was electroblotted onto poly(vinylidene difluoride) membrane. Ten randomly chosen spots were analyzed. With as few as three peptide masses, each protein was uniquely identified from over 91,000 protein sequences. All identifications were verified by concurrent N-terminal sequencing of identical spots from a second blot. One of the spots contained an N-terminally blocked protein that required enzymatic cleavage, peptide separation, and Edman degradation for confirmation of its identity.