Efficient in‐gel digestion procedure using 5‐cyclohexyl‐1‐pentyl‐β‐D‐maltoside as an additive for gel‐based membrane proteomics

Abstract

A cycloalkyl aliphatic saccharide, 5‐cyclohexyl‐1‐pentyl‐β‐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 © 2004 John Wiley & Sons, Ltd.