Identification of peanut and hazelnut allergens by native two-dimensional gel electrophoresis.
A procedure for the native two-dimensional electrophoresis of peanut and hazelnut proteins is described. Proteins were solubilised after acetone treatment using a combination of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and tetramethylene sulphone. These extracts were analysed by a combination of isoelectric focusing in the presence of lactose in immobilized pH gradients followed by charge shift electrophoresis. Immunoblot analysis, using sera from nut allergic patients, allowed the identification of a peanut and hazelnut allergen with identical isoelectric point and apparent molecular mass. These proteins were recovered from duplicate gels using a mixture of formic acid, acetonitrile (ACN) and isopropanol. The molecular masses for both proteins, determined by matrix assisted laser desorption/ionisation-mass spectrometry (MALDI-MS), were 4826 Da.
Hird
H
H
Central Science Laboratory, Sand Hutton, York, UK.
Pumphrey
R
R
Wilson
P
P
Sunderland
J
J
Reece
P
P
D019840:2-Propanol; D000097:Acetonitriles; D000485:Allergens / Q000032:analysis* / Q000302:isolation & purification; D010367:Arachis / Q000276:immunology*; D015153:Blotting, Western; D002793:Cholic Acids; D003902:Detergents; D015180:Electrophoresis, Gel, Two-Dimensional; D005512:Food Hypersensitivity / Q000097:blood / Q000276:immunology; D005561:Formates; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008970:Molecular Weight; D009754:Nuts / Q000276:immunology*; D010936:Plant Extracts / Q000737:chemistry / Q000276:immunology; D010940:Plant Proteins / Q000032:analysis* / Q000302:isolation & purification; D011852:Radioallergosorbent Test; D012995:Solubility; D012997:Solvents; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000097:Acetonitriles; D000485:Allergens; D002793:Cholic Acids; D003902:Detergents; D005561:Formates; D010936:Plant Extracts; D010940:Plant Proteins; D012997:Solvents; C030544:formic acid; D007073:Immunoglobulin E; D019840:2-Propanol; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; C032159:acetonitrile