Hirano H, Komatsu S, Takakura H, Sakiyama F, Tsunasawa S
Institute for Protein Research, Osaka University.
J Biochem. 1992 Jun;111(6):754-7. doi: 10.1093/oxfordjournals.jbchem.a123831.
A method was developed for direct microsequencing of N alpha-acetylated proteins electroblotted onto polyvinylidene difluoride membranes from polyacrylamide gels. N alpha-Acetylated proteins (greater than 32 pmol), including horse heart cytochrome c, five mutants of yeast cytochrome c, and bovine erythrocyte superoxide dismutase, were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. The portions of the membrane carrying the bands were cut out and treated with 0.5% polyvinylpyrrolidone in acetic acid solution at 37 degrees C for 30 min. The protein was digested on the membrane with 5-10 micrograms of trypsin at 37 degrees C for 24 h. During tryptic digestion, the resultant peptides were released from the membrane and the N-terminal peptide was efficiently deblocked with 50 mU of acylamino acid-releasing enzyme at 37 degrees C for 12 h. Picomole levels of the deblocked proteins could be sequenced directly by use of a gas-phase protein sequencer.
开发了一种直接对从聚丙烯酰胺凝胶电印迹到聚偏二氟乙烯膜上的Nα-乙酰化蛋白质进行微量测序的方法。Nα-乙酰化蛋白质（大于32皮摩尔），包括马心细胞色素c、酵母细胞色素c的五个突变体和牛红细胞超氧化物歧化酶，通过SDS-PAGE分离并电印迹到聚偏二氟乙烯膜上。将带有条带的膜部分切下，在37℃下用0.5%聚乙烯吡咯烷酮的乙酸溶液处理30分钟。在膜上用5-10微克胰蛋白酶在37℃下消化蛋白质24小时。在胰蛋白酶消化过程中，产生的肽从膜上释放出来，并且N端肽在37℃下用50毫微单位的酰基氨基酸释放酶有效去封闭12小时。去封闭的蛋白质的皮摩尔水平可以通过使用气相蛋白质测序仪直接测序。
