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Rapid Commun Mass Spectrom. 2004;18(20):2388-94.

Efficient in-gel digestion procedure using 5-cyclohexyl-1-pentyl-beta-D-maltoside as an additive for gel-based membrane proteomics.

Author information

1
Laboratory of Seeds Finding Technology, Eisai Co. Ltd., Tokodai 5-1-3, Tsukuba, Ibaraki 300-2635, Japan.

Abstract

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.

PMID:
15386632
DOI:
10.1002/rcm.1637
[Indexed for MEDLINE]

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