INTERNAL AMINO-ACID SEQUENCE-ANALYSIS OF PROTEINS SEPARATED BY ONE-DIMENSIONAL OR TWO-DIMENSIONAL GEL-ELECTROPHORESIS AFTER INSITU PROTEASE DIGESTION ON NITROCELLULOSE
INTERNAL AMINO-ACID SEQUENCE-ANALYSIS OF PROTEINS SEPARATED BY ONE-DIMENSIONAL OR TWO-DIMENSIONAL GEL-ELECTROPHORESIS AFTER INSITU PROTEASE DIGESTION ON NITROCELLULOSE
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DOI：
10.1073/pnas.84.20.6970
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发表时间：
1987-10-01
影响因子：
11.1
通讯作者：
KENT, SBH
中科院分区：
文献类型：
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作者：
AEBERSOLD, RH;LEAVITT, J;KENT, SBH
我们发展了一种通用的两步法从凝胶电泳法分离的皮摩尔数量的蛋白质中获得用于序列分析的多肽片段。蛋白质经一维或二维聚丙烯酰胺凝胶电泳法分离后，电泳法转移(电印迹)到硝基纤维素上，用可逆染色法检测并切下含蛋白质的区域，每种蛋白质被胰酶或葡萄球菌V-8蛋白酶等蛋白水解酶原位消化。得到的多肽片段用窄口径反相高效液相色谱分离，收集，并在气相测序仪中进行测序。良好的多肽回收率和在多肽片段混合物的分离中没有外来污染物，使得可以从皮摩尔量的蛋白质中产生广泛的内部序列信息。因此，该方法克服了从N末端封闭的蛋白质中获得氨基酸序列数据的问题，并提供了多个独立的序列延伸段，其可用于产生用于分子克隆的寡核苷酸探针和/或用于搜索相关蛋白质的序列数据库。该方法已成功地应用于从一维和二维聚丙烯酰胺凝胶中分离出的多种蛋白质的常规氨基酸序列分析。
We have developed a general two-step method for obtaining peptide fragments for sequence analysis from picomole quantities of proteins separated by gel electrophoresis. After separation by one- or two-dimensional polyacrylamide gel electrophoresis, proteins are electrophoretically transferred (electroblotted) onto nitrocellulose, the protein-containing regions are detected by reversible staining and are cut out, and each protein is digested in situ by proteolytic enzymes such as trypsin or staphylococcal V-8 protease. The resulting peptide fragments are separated by narrow-bore reverse-phase HPLC, collected, and sequenced in a gas-phase sequenator. Excellent peptide recoveries and the absence of extraneous contaminants in the separation of the peptide fragment mixture allow the generation of extensive internal sequence information from picomole amounts of protein. The method thus overcomes the problem of obtaining amino acid sequence data from N-terminally blocked proteins and provides multiple, independent stretches of sequence that can be used to generate oligonucleotide probes for molecular cloning and/or used to search sequence data bases for related proteins. This method has been successfully applied to the routine amino acid sequence analysis of a wide range of proteins isolated from one- and two-dimensional polyacrylamide gels.