TY - JOUR
T1 - Efficient in-gel digestion procedure using 5-cyclohexyl-1-pentyl-β-D- maltoside as an additive for gel-based membrane proteomics
AU - Katayama, Hiroyuki
AU - Tabata, Tsuyoshi
AU - Ishihama, Yasushi
AU - Sato, Toshitaka
AU - Oda, Yoshiya
AU - Nagasu, Takeshi
PY - 2004
Y1 - 2004
N2 - A cycloalkyl aliphatic saccharide, 5-cyclohexyl-5-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 0.5mm 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.
AB - A cycloalkyl aliphatic saccharide, 5-cyclohexyl-5-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 0.5mm 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.
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U2 - 10.1002/rcm.1637
DO - 10.1002/rcm.1637
M3 - Article
C2 - 15386632
AN - SCOPUS:5444256180
SN - 0951-4198
VL - 18
SP - 2388
EP - 2394
JO - Rapid Communications in Mass Spectrometry
JF - Rapid Communications in Mass Spectrometry
IS - 20
ER -