High-temperature protein mass mapping using a thermophilic protease
High-temperature protein mass mapping using a thermophilic protease
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DOI：
10.1021/ja002909n
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发表时间：
2001-02-28
影响因子：
15
通讯作者：
Siuzdak, G
中科院分区：
文献类型：
--
作者：
Bark, SJ;Muster, N;Siuzdak, G
基因组学的革命最近产生了整个基因组的大量序列数据库。蛋白质组学是研究由这些DNA序列编码的蛋白质。蛋白质组学分析的主要技术包括2-D凝胶电泳，然后是蛋白水解消化，质谱和计算机辅助数据分析。1通过酶促降解产生的靶蛋白的蛋白水解片段的质量可以直接以高准确度测量（对于1000 Da肽为0.005 Da）。或者，液相色谱串联质谱法可用于分析蛋白质消化片段。然后将该信息与数据库中蛋白质预测的理论蛋白水解片段进行比较，并对匹配进行统计评估。这种策略的成功依赖于数据库中蛋白质序列的存在，但随着整个基因组序列的完成，匹配的可能性很高，这种方法的两个限制是充分蛋白质水解所需的时间和某些蛋白质对消化的抗性。用胰蛋白酶充分消化通常需要4-24小时。此外，许多紧凑的球状蛋白质表现出对蛋白酶切割的抗性，因此不能通过可用的方法进行有效分析。这些蛋白质通常是具有关键生物功能的较大蛋白质复合物的组成部分。影响蛋白质水解的主要因素是蛋白质结构的移动性，这是一个受化学变性剂或共溶剂影响的过程。或者，高温也具有增加柔性的效果，因此增加蛋白质的蛋白水解敏感性。3这种热变性过程的优点是不需要额外的样品制备或纯化。我们利用热变性来产生一种简单的蛋白质水解方法来鉴定蛋白质。这种方法的关键是使用一种嗜热酶，嗜热菌蛋白酶，在高温下表现出最佳活性。4
The revolution of genomics has recently generated massive sequence databases for entire genomes. Proteomics is the study of the proteins encoded by these DNA sequences. The primary techniques for proteomic analysis consist of 2-D gel electrophoresis followed by proteolytic digestion, mass spectrometry, and computer-facilitated data analysis. 1 Masses for proteolytic fragments of the target protein generated by enzymatic degradation can be measured directly with high accuracy ((0.005 Da for a 1000 Da peptide). Alternatively, liquid chromatography tandem mass spectrometry can be used to analyze the protein digest fragments. This information is then compared with the theoretical proteolytic fragments predicted for proteins within a database, and matches are statistically evaluated. The success of this strategy relies on the existence of the protein sequence within the database, but with the sequences of whole genomes being completed, the likelihood for matches is high.Two limitations of this method are the time required for adequate proteolysis and the resistance of some proteins to digestion. Adequate digestion with trypsin typically requires 4-24 h. In addition, many compact globular proteins exhibit resistance to cleavage by proteases and, thus, cannot be effectively analyzed by available procedures. These proteins are often constituents in larger protein complexes with critical biological functions. The primary factor affecting the proteolysis of proteins is the mobility of protein structure, 2 a process influenced by chemical denaturants or cosolvents. Alternatively, high temperature also has the effect of increasing flexibility and, therefore, the proteolytic susceptibility of a protein. 3 This thermal denaturation process has the advantage of requiring no extra sample preparation or purification. We have taken advantage of thermal denaturation to generate a facile proteolysis method for identifying proteins. The key to this method is the use of a thermophilic enzyme, thermolysin, that exhibits optimal activity at elevated temperatures. 4