title: Investigating the applicability of antibodies generated within the human protein atlas as capture agents in immunoenrichment coupled to mass spectrometry.
abstract: For identification and characterization of proteins in complex samples, immunoenrichment coupled to mass spectrometry is a good alternative due to the sensitivity of the affinity enrichment and the specificity of mass spectrometry analysis. Antibodies are commonly used affinity agents; however, for high-throughput analysis, antibody availability is usually a bottleneck. Here we present a protocol for immunoenrichment coupled to mass spectrometry in a high-throughput setup, where all steps from bead coupling to mass spectrometry sample preparation are performed in parallel in a 96-well format. Antibodies generated within the Human Protein Atlas project were tested for applicability as capture agents. The antibodies were covalently attached to protein A beads, making it possible to reuse the coupled beads at least three times without destroying the antibody binding efficiency. Target proteins were captured from a U251 MG cell lysate, eluted, digested, and analyzed using mass spectrometry. Of 30 investigated antibodies, around 50% could successfully capture the corresponding native target protein, making the available library of more than 21 000 antibodies a valuable resource for immunoenrichment assays. Due to the diversity of different antibodies regarding affinity and specificity, analyzing antibodies in a high-throughput format is challenging. Even though protocol optimization for individual antibodies can be advantageous for future studies, our method enables a fast screening strategy to determine the usefulness of antibodies in immunoenrichment setups. In addition, we show that the specificity of the antibodies can be investigated by using label-free quantification.
authors.affiliation: Department of Protein Technology, KTH-Royal Institute of Technology , SE-106 91 Stockholm, Sweden.
authors.forename: Tove
authors.initials: T
authors.lastname: Boström
authors.forename: Henrik J
authors.initials: HJ
authors.lastname: Johansson
authors.forename: Janne
authors.initials: J
authors.lastname: Lehtiö
authors.forename: Mathias
authors.initials: M
authors.lastname: Uhlén
authors.forename: Sophia
authors.initials: S
authors.lastname: Hober
mesh_terms: D000906:Antibodies / Q000276:immunology*; D015153:Blotting, Western; D057166:High-Throughput Screening Assays; D006801:Humans; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D011506:Proteins / Q000276:immunology*; D020543:Proteome
chemical_list: D000906:Antibodies; D011506:Proteins; D020543:Proteome
keywords: MS; antibody enrichment; antibody specificity; antibody validation; immunoenrichment; label-free quantification; mass spectrometry