title: Derivatized nanoparticle coated capillaries for purification and micro-extraction of proteins and peptides.
abstract: Various methods are used to enrich or purify a protein of interest from other proteins and components in a crude cell lysate or other sample. One of the most powerful methods is affinity purification, also called affinity chromatography, whereby the proteins of interest are purified by virtue of their specific binding properties to an immobilized ligand. Affinity purification is becoming more widely used for exploring post-translation modifications and protein-protein interactions, especially with a view toward developing new general tag systems and strategies of chemical derivatization on peptides for affinity selection. Our work was aimed to immobilize proteins or ligands for affinity purification of antibodies, fusion-tagged proteins and other proteins and peptides. Selected proteins or peptides are efficiently extracted and enriched using chemically derivatized walls of a fused silica capillary column. In this paper, we present an open tubular capillary, where the inner wall of a fused silica capillary was derivatized by covalent binding of modified polystyrene latex particles. The capillaries were derivatized with iminodiacetic acid and loaded with Fe3+ or Ni2+ for the purification and enrichment of phosphopeptides or His-tagged proteins, respectively. The latex coated capillaries have been successfully applied to enrich phosphopeptides from beta-casein tryptic digest and ovalbumin tryptic digest at a micro volume scale with recoveries ranging from 92 to 95%. The capillaries have been eluted under conditions compatible with MALDI-MS without any prior desalting step. In another approach, concanavalin A (Con A) or Protein G were immobilized on the epoxy modified latex on the inner wall of the fused silica capillary for the purification of glycoproteins and immunoglobulin, respectively. The design of the capillary and the protocols used for purification permits the direct detection of eluted proteins and peptides with gel electrophoresis or with mass spectrometry. The elution volumes are passed as discrete segments of few microliters over the inner surface of the open-tube capillary, achieving enrichment factors of more than 20-fold from starting samples.
authors.affiliation: Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innrain 52a, 6020 Innsbruck, Austria. rania.bakry@uibk.ac.at
authors.forename: R
authors.initials: R
authors.lastname: Bakry
authors.forename: D
authors.initials: D
authors.lastname: Gjerde
authors.forename: G K
authors.initials: GK
authors.lastname: Bonn
mesh_terms: D002364:Caseins / Q000032:analysis; D002846:Chromatography, Affinity; D003208:Concanavalin A / Q000032:analysis; D007501:Iron / Q000737:chemistry; D049329:Nanostructures / Q000737:chemistry*; D009419:Nerve Tissue Proteins / Q000032:analysis; D009532:Nickel / Q000737:chemistry; D010047:Ovalbumin / Q000032:analysis; D010748:Phosphopeptides / Q000032:analysis*; D011137:Polystyrenes / Q000737:chemistry; D011485:Protein Binding; D011506:Proteins / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D002364:Caseins; C029152:G-substrate; D009419:Nerve Tissue Proteins; D010748:Phosphopeptides; D011137:Polystyrenes; D011506:Proteins; D003208:Concanavalin A; D009532:Nickel; D010047:Ovalbumin; D007501:Iron