Hirano H, Komatsu S, Kajiwara H, Takagi Y, Tsunasawa S
National Institute of Agrobiological Resources, Ibaraki, Japan.
Electrophoresis. 1993 Sep;14(9):839-46. doi: 10.1002/elps.11501401134.
A technique has been developed for efficient deblocking and subsequent microsequencing of N-terminally blocked proteins immobilized on a polyvinylidene difluoride (PVDF) membrane at the picomole levels. In this technique, proteins were first separated by polyacrylamide gel electrophoresis and then transferred onto a PVDF membrane by Western blotting. The electroblotted proteins with N-terminal acetylserine or acetylthreonine could be deblocked on-membrane by treatment with trifluoroacetic acid vapor and sequenced by a gas-phase protein sequencer. Similarly, N-formylated proteins could be deblocked on-membrane in HC1 solution and then directly sequenced from the N-terminal amino acid. Proteins with N-terminal pyroglutamic acid were enzymatically deblocked by in situ pyroglutamyl peptidase digestion, and N-acetylated proteins were also enzymatically deblocked with acylamino acid-releasing enzyme (AARE) after on-membrane digestion with trypsin to generate the N-terminal peptide fragment. This tryptic digestion was required since AARE can remove the acetylamino acid only from a short peptide. Based on these four deblocking methods, we present a strategy for sequential deblocking and subsequent N-terminal sequence analysis of N-blocked protein immobilized on PVDF membrane.
已开发出一种技术，可对皮摩尔水平固定在聚偏二氟乙烯（PVDF）膜上的N端封闭蛋白进行高效去封闭及后续微量测序。在该技术中，蛋白质首先通过聚丙烯酰胺凝胶电泳分离，然后通过蛋白质印迹法转移到PVDF膜上。带有N端乙酰丝氨酸或乙酰苏氨酸的电印迹蛋白可通过用三氟乙酸蒸汽处理在膜上去封闭，并通过气相蛋白质测序仪进行测序。同样，N-甲酰化蛋白可在HCl溶液中在膜上去封闭，然后直接从N端氨基酸开始测序。带有N端焦谷氨酸的蛋白质通过原位焦谷氨酰肽酶消化进行酶促去封闭，N-乙酰化蛋白在用胰蛋白酶进行膜上消化以产生N端肽片段后，也用酰基氨基酸释放酶（AARE）进行酶促去封闭。由于AARE只能从短肽中去除乙酰氨基酸，因此需要进行这种胰蛋白酶消化。基于这四种去封闭方法，我们提出了一种对固定在PVDF膜上的N封闭蛋白进行顺序去封闭及后续N端序列分析的策略。
