Elucidation of the primary structures of proteins by mass spectrometry.
Elucidation of the primary structures of proteins by mass spectrometry.
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通过质谱法阐明蛋白质的一级结构。
DOI：
10.1016/0003-2697(91)90050-4
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发表时间：
1991
影响因子：
2.9
通讯作者：
Green,B
中科院分区：
文献类型：
--
作者：
Smith,JB;Thévenon-Emeric,G;Smith,DL;Green,B
描述了一种新的质谱方法的组合，该方法可用于确定蛋白质的一级结构，包括翻译后修饰，具有前所未有的速度和准确性。牛晶状体α-晶体蛋白的结构表征已被用于说明该方法。通过反相HPLC法测定分馏但未达到均匀度的α-晶状体蛋白的分子量，不确定度为0.01%，其准确度至少是使用常规方法的100倍。该信息用于鉴定初级基因产物以及其磷酸化和截短形式。这些蛋白质的蛋白水解酶的分子量图，确定了直接耦合毛细管HPLC快原子轰击质谱。从这些图谱中，确认了整个氨基酸序列，并鉴定了磷酸化肽。磷酸化肽的MS/MS子离子质谱提供了足够的信息来确定哪个残基被磷酸化。由于蛋白质结构，包括翻译后修饰，是根据分子量确定的，这种方法具有广泛的应用，并将用于蛋白质结构解析中的各种各样的和具有挑战性的问题。
A combination of new mass spectrometric methods that can be used to determine the primary structures of proteins, including post-translational modifications, with unprecedented speed and accuracy is described. Structural characterization of α-crystallins from bovine lenses has been used to illustrate the methods. The molecular weights of α-crystallins fractionated, but not to homogeneity, by reversed-phase HPLC were determined with an uncertainty of 0.01% which is at least 100 times more accurate than is possible using conventional methods. This information was used to identify the primary gene product as well as its phosphorylated and truncated forms. Molecular weight maps of proteolytic digests of these proteins were determined by directly coupled capillary HPLC fast atom bombardment-mass spectrometry. From these maps, the entire amino acid sequence was confirmed, and the phosphorylated peptide was identified. The MS/MS daughter ion mass spectrum of the phosphorylated peptide provided sufficient information to determine which residue was phosphorylated. Because protein structure, including post-translational modifications, is determined on the basis of molecular weight, this method has broad application and will be useful for a variety of diverse and challenging problems in protein structure elucidation.