The Holdup Multiplex, an assay for high-throughput measurement of protein-ligand affinity constants using a mass-spectrometry readout

Abstract

The accurate description and subsequent modeling of protein interactomes requires quantification of their affinities at proteome-wide scale. Here we develop and validate the Holdup Multiplex, a versatile assay for high-throughput measurement of protein-ligand affinity constants that uses mass-spectrometry as readout. The method can quantify thousands of affinities in one single run, with high precision and over several orders of magnitude. We applied this strategy to the seven human 14 3 3 isoforms, quantifying in a few sample-runs their interaction with 1,000 different phosphopeptides. We were able to identify hundreds of new 14-3-3 binding sites. We showed that the seven human 14-3-3 display similar specificities but staggered affinities, 14-3-3γ being always the best binder and 14-3-3ϵ and σ, the weakest. Finally, we identified dozens of 14-3-3 bindings sites, some intervening in key signaling pathways, that were either stabilized or destabilized by the phytotoxin Fusicoccin-A. Our approach, which throughput can be pushed up to the sensitivity limit of the mass-spectrometry set-up, is applicable to any category of protein-ligand interactions and thus bears a wide potential both for high-throughput interactomics and chemoproteomics.

Uncovering the BIN1-SH3 interactome underpinning centronuclear myopathy

“…The main advantage of holdup over conventional pulldown-based approaches is that it captures the undisturbed binding equilibrium by quantifying the relative amount of unbound preys in the supernatant, instead of measuring the enrichment of bound prey on the resin after washing steps (Charbonnier et al , 2006). These measured relative prey concentrations, often referred to as binding intensities (BI), can be converted to equilibrium dissociation constants (Vincentelli et al , 2015) (Gógl et al , 2019) (Gogl et al , 2020) (Delalande et al , 2022). A recent variation of the method, called native holdup (nHU), uses dilute cell extracts as analyte, providing estimates of equilibrium dissociation constants for thousands of full-length endogenous proteins from a single experiment (Zambo et al , 2022).…”

“…The main advantage of holdup over conventional pulldown-based approaches, such as immunoprecipitation, is that it captures the undisturbed binding equilibrium allowing the determination of steady-state binding constants, instead of measuring the enrichment of bound prey on the resin after washing steps that only allows qualitative assessment of binding (Charbonnier et al, 2006). The measured relative prey depletion, often referred to as binding intensities (BI), can be converted to equilibrium dissociation constants (Delalande et al, 2022; Gogl et al, 2020; Gógl et al, 2019; Vincentelli et al, 2015). For this assay, we used streptavidin resin saturated with either biotin (control) or a synthetic biotinylated peptide derived from the C-terminal PRR region of DNM2 (residues 823-860) as a bait.…”

“…The main advantage of holdup over conventional pulldown-based approaches, such as immunoprecipitation, is that it captures the undisturbed binding equilibrium by quantifying the relative amount of unbound preys in the supernatant, instead of measuring the enrichment of bound prey on the resin after washing steps (Charbonnier et al, 2006). These measured relative prey concentrations, often referred to as binding intensities (BI), can be converted to equilibrium dissociation constants (Delalande et al, 2022; Gogl et al, 2020; Gógl et al, 2019; Vincentelli et al, 2015). We selected one common, likely benign (T532M), and eight rare variants with unknown clinical significance (Y531S, D537V, Q540H, P551L, V566M, R581C, V583I, and F584S) of the BIN1 SH3 domain from genomic databases, such as dbSNP, ClinVar, or gnomAD (Karczewski et al, 2020; Landrum et al, 2020; Sherry et al, 2001).…”

Molecules interact. But how strong and how much?

“…[34] Although their goals went unfinished, others proceeded with similar goals and measured (relative or absolute) affinities of large interaction networks of domain-motif interactions. [27,[35][36][37][38][39] Scientists today have access to dozens of orthogonal experimental approaches that can quantify affinity interactomes at proteomic scales of either minimal binding fragments or even full-length proteins, but mainstream interactomics still remains mostly qualitative.…”