Biotin-transfer from a trifunctional crosslinker for identification of cell surface receptors of soluble protein ligands

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DOIResolve DOI: https://doi.org/10.1038/srep46574
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  1. National Research Council of Canada. Human Health Therapeutics
FormatText, Article
Journal titleScientific Reports
ISSN2045-2322
2045-2322
Volume7
Article number46574
Pages# of pages: 9
Abstract
Here we describe a novel crosslinker and its application as a biotin-transfer reagent to identify cell surface receptors of soluble protein ligands on live cells. This crosslinker contains three functional groups: an aldehyde-reactive aminooxy group, a sulfhydryl, and a biotin (ASB). It is readily synthesized via a 3-step addition reaction using standard solid-phase peptide synthesis methods and commercially available intermediates, allowing access to laboratories without specialized synthetic chemistry capabilities. For the biotin-transfer method, ASB is linked to a protein ligand through the sulfhydryl group in a two-step process that allows the introduction of a disulfide bond between the ligand and the crosslinker. Incubation of the labelled ligand with oxidized live cells leads to the formation of crosslinks with aldehyde-containing glycans on the cell surface receptor. Subsequent reduction of the disulfide bond results in biotin transfer from the ligand to the cell surface receptor. Protein biotinylation that is mediated by ligand binding to its receptor is differentiated from background biotinylation events by comparison with a similarly labelled control protein using comparative proteomic mass spectrometry to quantify streptavidin-bound proteins. Using this method, we successfully identified the cell surface receptors of a peptide hormone, a monoclonal antibody, and a single-domain antibody-Fc fusion construct.
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PublisherNature Publishing Group
LanguageEnglish
Peer reviewedYes
NPARC number23002205
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Record identifier7966c94a-3e77-45fa-898d-5aab767da2f8
Record created2017-09-05
Record modified2020-05-30
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