Ligand-based receptor identification on living cells and tissues using TRICEPS

Abstract

Physiological responses to ligands such as peptides, proteins, pharmaceutical drugs or whole pathogens are generally mediated through interactions with specific cell surface protein receptors. Here we describe the application of TRICEPS, a specifically designed chemoproteomic reagent that can be coupled to a ligand of interest for the subsequent ligand-based capture of corresponding receptors on living cells and tissues. This is achieved by three orthogonal functionalities in TRICEPS-one that enables conjugation to an amino group containing ligands, a second for the ligand-based capture of glycosylated receptors on gently oxidized living cells and a biotin tag for purifying receptor peptides for analysis by quantitative mass spectrometry (MS). Specific receptors for the ligand of interest are identified through quantitative comparison of the identified peptides with a sample generated by a control probe with known (e.g., insulin) or no binding preferences (e.g., TRICEPS quenched with glycine). In combination with powerful statistical models, this ligand-based receptor capture (LRC) technology enables the unbiased and sensitive identification of one or several specific receptors for a given ligand under near-physiological conditions and without the need for genetic manipulations. LRC has been designed for applications with proteins but can easily be adapted for ligands ranging from peptides to intact viruses. In experiments with small ligands that bind to receptors with comparatively large extracellular domains, LRC can also reveal approximate ligand-binding sites owing to the defined spacer length of TRICEPS. Provided that sufficient quantities of the ligand and target cells are available, LRC can be carried out within 1 week.

Laminin targeting of a peripheral nerve-highlighting peptide enables degenerated nerve visualization

“…No specific protein was selectively captured as determined by MS analysis, leading us to speculate that NP41 bound with low affinity to homogenates. Thus, to capture NP41-receptor interaction in situ, we developed a method for proximity labeling ligand-bound proteins using photooxidation and targeted BH labeling and also tested previously described glycoprotein capture using TRICEPS-based chemistry (8,9).…”

“…A variety of methods to capture native ligand-receptor interactions have used chemical or photo-cross-linking followed by mass spectrometry (MS), some requiring that ligands retain binding activity after potentially disruptive chemical treatment (6)(7)(8)(9). In most cross-linking techniques, the ligand must reach an appropriately reactive site on the target for cross-linking to occur, while being conjugated to a potentially bulky purification tag, which can weaken specific binding.…”

“…Alternatively, ligand-directed receptor capture (LRC) using TRICEPS technology has been shown to enable carbohydratedirected tagging of receptors and subsequent identification by MS (8,9). Mild sodium periodate (NaIO 4 ) treatment oxidizes cisglycol groups in carbohydrates to aldehydes, and newly generated aldehydes cross-link to a BH-containing ligand.…”

Serum stimulation of CCR7 chemotaxis due to coagulation factor XIIa-dependent production of high-molecular-weight kininogen domain 5

“…The TriCEPS reagent is a proprietary trifunctional molecule with three key moieties: (i) an amine-reactive group capable of binding a peptide ligand of interest, (ii) a cross-linking group capable of bonding to oxidized glycans, and (iii) an affinity tag for downstream extraction (20,50). These studies were performed by DualSystems Biotech.…”

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

“…have recently shown that ligand-directed crosslinking using their TRICEPS crosslinker is an effective method for the identification of cell surface binding partners for soluble protein ligands23. In the TRICEPS method, soluble ligands are labelled with the trifunctional TRICEPS crosslinker through an amine reactive group and crosslinks are formed following binding of the labelled ligand to oxidized cells or tissues via hydrazone bond formation between a protected hydrazide group on the TRICEPS crosslinker and aldehyde-containing glycans on the cell surface receptor45.…”