Walker J E, Fearnley I M, Blows R A
Biochem J. 1986 Jul 1;237(1):73-84. doi: 10.1042/bj2370073.
A solid-phase protein microsequencer is described that has been designed to determine protein sequences with subnanomolar quantities of protein. Its utility has been demonstrated by the determination of many sequences in subunits of mitochondrial F1-ATPase, in a protein isolated from mouse gap junctions and in the mitochondrial phosphate-transporter protein. It has a number of advantages over liquid- and gas-phase sequencers. Firstly, the degradation cycle takes 24 min, more than twice as fast as any other sequencer. This helps to reduce exposure of proteins to inimical reagents and increases throughput of samples. Secondly, polar amino acids such as phosphoserine, and polar derivatives formed by active-site photoaffinity labelling with 8-azido-ATP, are recovered quantitatively from the reaction column and can be positively identified. In other types of sequencer these polar derivatives, being somewhat insoluble in butyl chloride, tend to remain in the reaction chamber of the instrument and so are more difficult to identify. The solid-phase protein sequencer is also more suited than the liquid-phase instrument for analysis of proteolipids from membranes. These hydrophobic proteins tend to dissolve in organic solvents during washing steps in the liquid-phase instrument and are lost. Covalent attachment as used in the solid-phase instrument solves this problem.
本文描述了一种固相蛋白质微量测序仪，其设计目的是用亚纳摩尔量的蛋白质来测定蛋白质序列。通过测定线粒体F1 - ATP酶亚基、从小鼠间隙连接中分离出的一种蛋白质以及线粒体磷酸盐转运蛋白中的许多序列，已证明了该仪器的实用性。与液相和气相测序仪相比，它具有许多优点。首先，降解循环需要24分钟，比任何其他测序仪快两倍多。这有助于减少蛋白质与有害试剂的接触，并提高样品通量。其次，极性氨基酸如磷酸丝氨酸，以及通过用8 - 叠氮基 - ATP进行活性位点光亲和标记形成的极性衍生物，可从反应柱中定量回收并能得到明确鉴定。在其他类型的测序仪中，这些极性衍生物在丁基氯中有些不溶，往往会留在仪器的反应室中，因此更难鉴定。固相蛋白质测序仪也比液相仪器更适合分析来自膜的蛋白脂质。在液相仪器的洗涤步骤中，这些疏水蛋白质往往会溶解在有机溶剂中而损失掉。固相仪器中使用的共价连接解决了这个问题。
