title: Gel-absorption-based sample preparation method for shotgun analysis of membrane proteome.
abstract: Membrane proteins solubilized in a starting buffer containing high concentration of sodium dodecyl sulfate (SDS) are directly entrapped and immobilized into gel matrix when the membrane protein solution is absorbed by the vacuum-dried polyacrylamide gel. After the detergent and other salts are removed by washing, the proteins are subjected to in-gel digestion and the tryptic peptides are extracted and analyzed by CapLC-MS/MS. The newly developed method not only avoids protein loss and the adverse protein modifications during gel-embedment but also improves the subsequent in-gel digestion and the recovery of tryptic peptides, particularly the hydrophobic peptides. Thus, this method facilitates the identification of membrane proteins especially the integral membrane proteins.
authors.affiliation: Hunan Normal University, Changsha, Hunan, People's Republic of China. wang_xianchun@263.net
authors.forename: Xianchun
authors.initials: X
authors.lastname: Wang
authors.forename: Songping
authors.initials: S
authors.lastname: Liang
mesh_terms: D000042:Absorption; D000180:Acrylic Resins / Q000737:chemistry; D000818:Animals; D002021:Buffers; D002462:Cell Membrane / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D008099:Liver / Q000737:chemistry / Q000166:cytology; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D010449:Peptide Mapping; D011489:Protein Denaturation; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry* / Q000302:isolation & purification; D051381:Rats; D019163:Reducing Agents / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry
chemical_list: D000180:Acrylic Resins; D002021:Buffers; D003902:Detergents; D008565:Membrane Proteins; D010446:Peptide Fragments; D020543:Proteome; D019163:Reducing Agents; C016680:polyacrylamide gels; D012967:Sodium Dodecyl Sulfate; D014357:Trypsin; D004229:Dithiothreitol