title: Proteomic analysis of erythrocyte membranes by soft Immobiline gels combined with differential protein extraction.
abstract: Improvements in proteome analysis of erythrocyte membrane proteins by two-dimensional electrophoresis are here reported. In particular, a differential extraction procedure was set up allowing separation of integral membrane proteins from peripheral species. Moreover, the use of dilute Immobiline gels (down to as low as 3% T matrix) permitted a better penetration and transfer inside the gel of proteins with large M(r). These protocol modifications, combined with sample delipidation and alkylation prior to electrophoresis, which prevented generation of homo- and hetero-oligomers following disulfide scrambling phenomena, allowed the display of more than 500 spots in the pI/M(r) plane. Among those, noteworthy was the presence of high levels of filamentous proteins, such as alpha-spectrin and ankyrins, or integral membrane proteins, such as band 3, band 4.1 and 4.2, not displayed or barely present in other maps exploiting immobilized pH gradients in the first dimension. Accordingly, our results show that this 2D mapping technique is a valuable tool in exploring pathologies related to genetic defects associated to membrane proteins.
authors.affiliation: Laboratory of Physiopathology of Uremia, G. Gaslini Children's Hospital, 16148 Genoa, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Bruschi
authors.forename: Claudio
authors.initials: C
authors.lastname: Seppi
authors.forename: Simona
authors.initials: S
authors.lastname: Arena
authors.forename: Luca
authors.initials: L
authors.lastname: Musante
authors.forename: Laura
authors.initials: L
authors.lastname: Santucci
authors.forename: Cesare
authors.initials: C
authors.lastname: Balduini
authors.forename: Andrea
authors.initials: A
authors.lastname: Scaloni
authors.forename: Marina
authors.initials: M
authors.lastname: Lanciotti
authors.forename: Pier Giorgio
authors.initials: PG
authors.lastname: Righetti
authors.forename: Giovanni
authors.initials: G
authors.lastname: Candiano
mesh_terms: D003902:Detergents / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000295:instrumentation / Q000379:methods; D004910:Erythrocyte Membrane / Q000737:chemistry*; D006801:Humans; D007526:Isoelectric Point; D008565:Membrane Proteins / Q000032:analysis / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020543:Proteome / Q000032:analysis*; D012972:Sodium Hydroxide / Q000737:chemistry
chemical_list: D003902:Detergents; D008565:Membrane Proteins; D020543:Proteome; D012972:Sodium Hydroxide