Efficient in-gel digestion procedure using 5-cyclohexyl-1-pentyl-β-D-maltoside as an additive for gel-based membrane proteomics
Efficient in-gel digestion procedure using 5-cyclohexyl-1-pentyl-β-D-maltoside as an additive for gel-based membrane proteomics
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DOI：
10.1002/rcm.1637
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发表时间：
2004-01-01
影响因子：
2
通讯作者：
Nagasu, T
中科院分区：
文献类型：
--
作者：
Katayama, H;Tabata, T;Nagasu, T
在96孔板高通量凝胶内蛋白质消化系统中，5-环己基-1-戊基-β-D-麦芽糖苷(Cymal-5)是一种新型的添加剂。在胰酶处理过程中添加0.1%的Cymal-5(最终浓度)，与基质辅助激光解吸电离飞行时间质谱仪(MALDI-TOFMS)和液相色谱/串联质谱仪(LC/MS/MS)分析都是兼容的，并且比我们先前报道的正辛基葡萄糖苷有更好的消化效率。半胱氨酸残基在变性条件下的凝胶内还原和烷基化也提高了多肽的序列覆盖率。用0.5 mm厚的凝胶、负染、变性条件下的烷基化(6M盐酸胍)和Cymal-5的最佳组合进行凝胶内胰酶消化，尤其是在膜蛋白分析中，疏水性多肽的回收率显著提高。该方法简便易行，可广泛应用于凝胶蛋白质组学。版权所有(C)2004 John Wiley Sons，Ltd.
A cycloalkyl aliphatic saccharide, 5-cyclohexyl-1-pentyl-beta-D-maltoside (CYMAL-5), was evaluated as a novel additive in a high-throughput in-gel protein digestion system using 96-well plates. Addition of 0.1% CYMAL-5 (final concentration) during trypsin treatment was compatible with both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis, and gave a better digestion efficiency than n-octylglucoside, which we previously reported. In-gel reduction and alkylation of Cys residues under denaturing conditions also improved the sequence coverage of peptides. In-gel tryptic digestion with the optimum combination of 0.5 mm thick gels, negative staining, alkylation under denaturing conditions (6 M guanidine hydrochloride), and digestion in the presence of CYMAL-5, gave excellent performance especially for membrane protein analysis, where recovery of hydrophobic peptides was markedly enhanced. The new protocol is simple and convenient, and should be widely applicable to gel-based proteomics. Copyright (C) 2004 John Wiley Sons, Ltd.