High affinity capture surface for matrix-assisted laser desorption/ionisation compatible protein microarrays.
A surface for the capture of biotin-tagged proteins on matrix-assisted laser desorption/ionisation (MALDI) targets has been investigated. Binding of a poly-L-lysine poly(ethylene glycol)-biotin polymer to glass and gold surfaces has been demonstrated using dual wavelength interferometry. Biotinylated proteins were captured onto this surface using tetrameric neutravidin as a multivalent bridging molecule. Biotin tagging of proteins was achieved by chemical biotinylation or by expressing a protein with a biotinylation consensus sequence in E. coli. The specificity of the surface for biotin-tagged proteins allowed the purification of biotin-tagged glutathione-S-transferase from a bacterial lysate directly onto a MALDI target. Subsequently, the protein was digested on the MALDI target and a protein fingerprint analysis confirmed its presence directly, but no E. coli proteins were detected. Therefore, we conclude that this surface is highly specific for the capture of biotin-labelled proteins and has low non-specific binding properties for non-biotinylated proteins. Furthermore, protein-protein interactions using biotinylated lectins were investigated, and the selective capture of the glycoprotein fetuin with wheat germ agglutinin was demonstrated. Also, immobilised Arachis hypogea agglutinin recognised a minor asialo component of this glycoprotein on the array. The high affinity immobilisation of proteins onto this surface allowed effective desalting procedures to be used which improved the desorption of high molecular weight proteins. Another aspect of this surface is that a highly ordered coupling of the analyte can be achieved which eliminates the search for the sweet spot and allows the creation of densely packed protein microarrays for use in mass spectrometry.
Koopmann
Jens-Oliver
JO
Procognia Ltd, Babraham Hall, Babraham, Cambridge CB2 4AT, UK. Jens.koopmann@procognia.com
Blackburn
Jonathan
J
D010367:Arachis / Q000737:chemistry; D001710:Biotin / Q000378:metabolism; D010449:Peptide Mapping; D011092:Polyethylene Glycols / Q000737:chemistry; D011107:Polylysine / Q000031:analogs & derivatives* / Q000737:chemistry; D040081:Protein Array Analysis / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation* / Q000379:methods; D014909:Wheat Germ Agglutinins / Q000737:chemistry; D000509:alpha-Fetoproteins / Q000032:analysis
D014909:Wheat Germ Agglutinins; D000509:alpha-Fetoproteins; C443714:polylysine-graft-(poly(ethylene glycol)); D011107:Polylysine; D011092:Polyethylene Glycols; D001710:Biotin