title: Advancing the global proteome survey platform by using an oriented single chain antibody fragment immobilization approach.
abstract: Increasing the understanding of a proteome and how its protein composition is affected by for example different diseases, such as cancer, has the potential to improve strategies for early diagnosis and therapeutics. The Global Proteome Survey or GPS is a method that combines mass spectrometry and affinity enrichment with the use of antibodies. The technology enables profiling of complex proteomes in a species independent manner. The sensitivity of GPS, and other methods relying on affinity enrichment, is largely affected by the activity of the exploited affinity reagent. We here present an improvement of the GPS platform by utilizing an antibody immobilization approach which ensures a controlled immobilization process of the antibody to the magnetic bead support. More specifically, we make use of an antibody format that enables site-directed biotinylation and use this in combination with streptavidin coated magnetic beads. The performance of the expanded GPS platform was evaluated by profiling yeast proteome samples. We demonstrate that the oriented antibody immobilization strategy increases the ability of the GPS platform and results in larger fraction of functional antibodies. Additionally, we show that this new antibody format enabled in-solution capture, i.e. immobilization of the antibodies after sample incubation. A workflow has been established that permit the use of an oriented immobilization strategy for the GPS platform.
authors.affiliation: Department of Immunotechnology, Lund University, Medicon Village (House 406), SE-223 81 Lund, Sweden.
authors.forename: Anna
authors.initials: A
authors.lastname: Säll
authors.affiliation: Department of Immunotechnology, Lund University, Medicon Village (House 406), SE-223 81 Lund, Sweden; Science for Life Laboratory, Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Helena
authors.initials: H
authors.lastname: Persson
authors.affiliation: Department of Immunotechnology, Lund University, Medicon Village (House 406), SE-223 81 Lund, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Ohlin
authors.affiliation: Department of Immunotechnology, Lund University, Medicon Village (House 406), SE-223 81 Lund, Sweden. Electronic address: Carl.Borrebaeck@immun.lth.se.
authors.forename: Carl A K
authors.initials: CA
authors.lastname: Borrebaeck
authors.affiliation: Department of Immunotechnology, Lund University, Medicon Village (House 406), SE-223 81 Lund, Sweden. Electronic address: Christer.Wingren@immun.lth.se.
authors.forename: Christer
authors.initials: C
authors.lastname: Wingren
mesh_terms: D000818:Animals; D055910:Antibodies, Immobilized; D001709:Biotechnology; D006801:Humans; D007128:Immunoglobulin Fragments; D018189:Immunomagnetic Separation; D051379:Mice; D040081:Protein Array Analysis; D020543:Proteome / Q000032:analysis* / Q000276:immunology*; D040901:Proteomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000276:immunology; D057127:Single-Chain Antibodies; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D055910:Antibodies, Immobilized; D007128:Immunoglobulin Fragments; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D057127:Single-Chain Antibodies