title: Improved in-gel approaches to generate peptide maps of integral membrane proteins with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
abstract: This paper reports studies of in-gel digestion procedures to generate MALDI-MS peptide maps of integral membrane proteins. The methods were developed for the membrane domain of the mannitol permease of E. coli. In-gel digestion of this domain with trypsin, followed by extraction of the peptides from the gel, yields only 44% sequence coverage. Since lysines and arginines are seldomly found in the membrane-spanning regions, complete tryptic cleavage will generate large hydrophobic fragments, many of which are poorly soluble and most likely contribute to the low sequence coverage. Addition of the detergent octyl-beta-glucopyranoside (OBG), at 0.1% concentration, to the extraction solvent increases the total number of peptides detected to at least 85% of the total protein sequence. OBG facilitates the recovery of hydrophobic peptides when they are SpeedVac dried during the extraction procedure. Many of the newly recovered peptides are partial cleavage products. This seems to be advantageous since it generates hydrophobic fragments with a hydrophilic solubilizing part. In-gel CNBr cleavage resulted in 5-10-fold more intense spectra, 83% sequence coverage, fully cleaved fragments and no effect of OBG. In contrast to tryptic cleavage sites, the CNBr cleavage sites are found in transmembrane segments; cleavage at these sites generates smaller hydrophobic fragments, which are more soluble and do not need OBG. With the results of both cleavages, a complete sequence coverage of the membrane domain of the mannitol permease of E. coli is obtained without the necessity of using HPLC separation. The protocols were applied to two other integral membrane proteins, which confirmed the general applicability of CNBr cleavage and the observed effects of OBG in peptide recovery after tryptic digestion.
authors.affiliation: Groningen Biomolecular Sciences and Biotechnology Institute and Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Bart A
authors.initials: BA
authors.lastname: van Montfort
authors.forename: Benito
authors.initials: B
authors.lastname: Canas
authors.forename: Ria
authors.initials: R
authors.lastname: Duurkens
authors.forename: Jasminka
authors.initials: J
authors.lastname: Godovac-Zimmermann
authors.forename: George T
authors.initials: GT
authors.lastname: Robillard
mesh_terms: D000595:Amino Acid Sequence; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D005960:Glucosides / Q000737:chemistry; D006868:Hydrolysis; D007202:Indicators and Reagents; D008565:Membrane Proteins / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010449:Peptide Mapping / Q000379:methods*; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry; D011492:Protein Hydrolysates / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D027981:Symporters; D014357:Trypsin
chemical_list: D029968:Escherichia coli Proteins; D005960:Glucosides; D007202:Indicators and Reagents; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011492:Protein Hydrolysates; D027981:Symporters; C018619:octyl-beta-D-glucoside; C021499:lactose permease; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli; D014357:Trypsin; D003488:Cyanogen Bromide