Fig. 1.

HATRIC-LRC enables ligand-based receptor capture. a Structure of hydrazone (highlighted in magenta) and azide (highlighted in turquoise) containing tri-functional compound HATRIC (NHS ester highlighted in blue). Mw = 1171.4 g mol⁻¹ (synthesis described in Supplementary Note 1). b Workflow of HATRIC-LRC for identification of target receptors of ligands on living cells. First, living cells are mildly oxidized with 1.5 mM NaIO4. HATRIC, conjugated to the ligand of interest, is added to living cells. The ligand selectively directs HATRIC to its glycoprotein target receptor, where HATRIC reacts to generate azide-tagged cell-surface glycoproteins catalyzed by 5-MA. In order to identify target receptors of orphan ligands, a dual track experimental setup is employed. In the control, the HATRIC-conjugated ligand is applied to the cells in the presence of an excess unmodified ligand. Alternatively, HATRIC can be quenched with glycine for a negative control or a ligand with known target receptors can be employed as a positive control (not depicted in figure). After lysis and affinity purification of azide-tagged proteins with unbound proteins removed by harsh washing, peptides are proteolyzed with trypsin. Peptides are identified with high-accuracy mass spectrometry in a data-dependent acquisition mode followed by quantitative comparison of peptide fractions? from experiment and control to reveal specific enrichment of candidate cell surface receptors. Target receptors are defined as proteins that have a fold change of >1.5 compared to the control as well as an FDR-adjusted p value (Benjamini–Hochberg method) equal to or smaller than 0.05, corresponding to a target receptor window in the volcano plot that is framed by dotted lines and highlighted in red. c Flow cytometry traces of U-2932 cells incubated with HATRIC conjugated to dibenzocyclooctyne-Alexa Fluor 488 (DIBO-AF488) at pH 6.5 or pH 7.4 in the presence or absence of organocatalyst 5-methoxyanthranilic acid (5-MA) (structure shown, Mw = 167.16 g mol⁻¹) or 2-amino-4,5-dimethoxy benzoic acid (ADA). HATRIC was quenched with glycine (Gly−) to avoid potential reaction of HATRIC’s NHS-ester with amino groups at the cell surface. Shift to the right indicates more efficient labeling with HATRIC-DIBO-AF488