Efficient in-gel digestion procedure using 5-cyclohexyl-1-pentyl-beta-D-maltoside as an additive for gel-based membrane proteomics.
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.
Katayama
Hiroyuki
H
Laboratory of Seeds Finding Technology, Eisai Co. Ltd., Tokodai 5-1-3, Tsukuba, Ibaraki 300-2635, Japan.
Tabata
Tsuyoshi
T
Ishihama
Yasushi
Y
Sato
Toshitaka
T
Oda
Yoshiya
Y
Nagasu
Takeshi
T
D000478:Alkylation; D001436:Bacteriorhodopsins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D045744:Cell Line, Tumor; D005591:Chemical Fractionation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003110:Colonic Neoplasms / Q000378:metabolism*; D005782:Gels / Q000737:chemistry; D005960:Glucosides / Q000737:chemistry*; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification*; D009363:Neoplasm Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D010741:Phospholipases A / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry
D005782:Gels; D005960:Glucosides; D008565:Membrane Proteins; D009363:Neoplasm Proteins; C400643:cyclohexyl-pentyl-maltoside; D001436:Bacteriorhodopsins; D010741:Phospholipases A; D014357:Trypsin