Nat. Biotechnol., published online 16 September 2012; doi:10.1038/nbt.2354


Identifying ligands for receptors in live cells can provide valuable information, providing insight into signaling pathways as well as drug targets, but these interactions can be difficult to detect and quantify. Frei et al. now report a trifunctional chemoproteomic reagent called TRICEPS that binds glycosylated receptors on live cells and allows for the purification of the ligand-receptor complex and identification of receptor-derived peptides by MS. TRICEPS contains three functional groups: an N-hydroxysuccinimide ester for ligand conjugation, a protected hydrazine that reacts with aldehydes introduced into the carbohydrates of cell-surface glycoproteins and biotin for the affinity capture of glycopeptides. The authors validated their approach by using insulin-coupled TRICEPS to isolate the insulin receptor from adipocytes. The authors then used their reagent to compare receptor interactions of EGF and trastuzumab, a therapeutic antibody that binds ErbB2, a member of the EGF receptor superfamily. The antibody bound selectively to ErbB2, whereas EGF bound EGFR; the preferential identification of only two glycosylated peptides in the EGF-EGFR samples provided insight into the ligand-binding site. Application of TRICEPS also revealed binding sites for DARPins, ankyrin repeat proteins, on ErbB2. Evaluation of trastuzumab-receptor interactions in primary cancer tissue revealed binding to ErbB2 as well as to immunoglobulin Fcγ receptors, indicating that trastuzumab can bind multiple targets in complex tissue. Taken together, these data indicate that TRICEPS allows ligand-based receptor capture from live cells and provides insight into the approximate binding site on receptors.