Journal: Scientific Reports
Article Title: Biotin-transfer from a trifunctional crosslinker for identification of cell surface receptors of soluble protein ligands
Figure Lengend Snippet: Biotin transfer workflow for identification of the ligand-binding partner. ( a) Ligand labelling: Ligands of interest are labelled in a two-step reaction through addition of a commercial amine-to-sulfhydryl crosslinker (LC-SPDP or PEG4 version) followed by addition of ASB. Both a control ligand and a ligand-of-interest are prepared in parallel. ( b ) Biotin-transfer to cell surface receptor on periodate-treated live cells: The labelled ligand in incubated with cells to encourage ligand-directed cell surface binding prior to addition of a catalyst to promote oxime bond formation and crosslinking. Cells are then solubilized under reducing conditions resulting in the transfer of the biotin from the ligand to its crosslinked binding partner. ( c ) Isolation of biotinylated proteins and identification of enriched peptides by comparative mass spectrometry: Biotinylated proteins are immobilized on a streptavidin resin and digested with trypsin. The intensity of peptides resulting from parallel experiments with the control ligand and the ligand-of-interest are then compared using standard mass spectrometry-based proteomic workflows.
Article Snippet: For the first step, 20 nmol of a linker, LC-SPDP or PEG4 -SPDP (Thermo Fisher), was incubated with the ligand for 30 minutes at RT.
Techniques: Ligand Binding Assay, Cell Surface Receptor Assay, Incubation, Binding Assay, Isolation, Mass Spectrometry