Advances in gas chromatographic mass spectrometric protein sequencing. 2--Application to membrane proteins.
Advances in gas chromatographic mass spectrometric protein sequencing. 2--Application to membrane proteins.
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气相色谱质谱蛋白质测序的进展。
DOI：
10.1002/bms.1200080204
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发表时间：
1981
期刊：
影响因子：
--
通讯作者：
Biemann,K
中科院分区：
文献类型：
--
作者：
Herlihy,WC;Anderegg,RJ;Biemann,K
的整体膜蛋白细菌视紫红质的一级结构，确定了一个有效的结合气相色谱质谱技术与埃德曼降解。这种方法的组合规避了许多与细菌视紫红质及其主要降解片段在水性缓冲液中的不溶性相关的实验困难。具体而言，在来自细菌视紫红质的溴化氰肽的气相色谱质谱分析中，已经可以鉴定作为构建C末端序列的起点的含高丝氨酸的肽。在大多数情况下，由气相色谱质谱肽构建的C-末端序列与Edman降解实验中获得的N-末端序列重叠，从而完成了片段的结构。此外，通过直接分析蛋白质片段部分水解产生的复杂混合物，实现了对建立溴化氰片段顺序所需的含甲硫氨酸肽的特异性鉴别。这些数据使得有可能确定的序列的大部分细菌视紫红质完全从溴化氰裂解，与这种疏水性蛋白质的溶解度特性兼容的少数特定反应之一。最后，气相色谱质谱序列数据已被用来分配或确认氨基酸的Edman数据是模糊的。这些气相色谱质谱技术可以有效且可靠地确定这种长为248个氨基酸的膜蛋白的完整序列。
The primary structure of the integral membrane protein bacteriorhodopsin was determined by an efficient combination of gas chromatographic mass spectrometric techniques with the Edman degradation. This combination of methodologies circumvented many of the experimental difficulties associated with the insolubility of bacteriorhodopsin and its primary degradation fragments in aqueous buffers. Specifically, in the gas chromatographic mass spectrometric analysis of the cyanogen bromide peptides derived from bacteriorhodopsin, it has been possible to identify homoserine‐containing peptides which served as a starting point for the construction of C‐terminal sequences. In most cases this C‐terminal sequence constructed from the gas chromatographic mass spectrometric peptides overlapped the N‐terminal sequence derived in an Edman degradation experiment, thereby completing the structure of the fragment. Furthermore, the specific identification of methionine‐containing peptides required to establish the order of the cyanogen bromide fragments was accomplished by direct analysis of the complex mixtures generated by partial hydrolysis of segments of the protein. These data made it possible to determine the sequence of a large portion of bacteriorhodopsin solely from cyanogen bromide cleavage, one of the few specific reactions compatible with the solubility properties of this hydrophobic protein. Finally, the gas chromatographic mass spectrometric sequence data have been used to assign or confirm amino acids where the Edman data was ambiguous. These gas chromatographic mass spectrometric techniques resulted in an efficient and reliable determination of the complete sequence of this membrane protein which is 248 amino acids long.