| Title: |
The Holdup Multiplex, an assay for high-throughput measurement of protein-ligand affinity constants using a mass-spectrometry readout |
| Authors: |
Delalande, François; Østergaard, So̷ren; Gógl, Gergő; Cousido-Siah, Alexandra; Mcewen, Alastair, G; Men, Yushi; Salimova, Farida; Rohrbacher, Aurélien; Kostmann, Camille; Nominé, Yves; Vincentelli, Renaud; Eberling, Pascal; Carapito, Christine; Travé, Gilles; Monsellier, Elodie |
| Contributors: |
Laboratoire de Spectrométrie de Masse BioOrganique Strasbourg (LSMBO); Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC); Institut Pluridisciplinaire Hubert Curien (IPHC); Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Institut Pluridisciplinaire Hubert Curien (IPHC); Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS); Novo Nordisk Inc; NOVO NORDISK PARK; Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC); Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS); Architecture et fonction des macromolécules biologiques (AFMB); Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE); Fédération de Recherche nationale ProFI (FR ProFI); Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS); ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010); ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010); ANR-10-IDEX-0002,UNISTRA,Par-delà les frontières, l'Université de Strasbourg(2010); ANR-20-SFRI-0012,STRAT'US,Façonner les talents en formation et en recherche à l'Université de Strasbourg(2020); ANR-17-EURE-0023,IMCBio,Integrative Molecular and Cellular Biology(2017) |
| Source: |
ISSN: 0002-7863. |
| Publisher Information: |
CCSD; American Chemical Society |
| Publication Year: |
2025 |
| Collection: |
HAL-CEA (Commissariat à l'énergie atomique et aux énergies alternatives) |
| Subject Terms: |
[SDV]Life Sciences [q-bio]; [CHIM]Chemical Sciences |
| Description: |
International audience ; 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, 14-3-3g 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 setup, is applicable to any category of protein-ligand interactions and thus bears a wide potential both for high-throughput interactomics and chemoproteomics. |
| Document Type: |
article in journal/newspaper |
| Language: |
English |
| Relation: |
BIORXIV: 2022.12.08.519103 |
| DOI: |
10.1101/2022.12.08.519103 |
| Availability: |
https://hal.science/hal-04246952; https://hal.science/hal-04246952v1/document; https://hal.science/hal-04246952v1/file/2022.12.08.519103v1.full.pdf; https://doi.org/10.1101/2022.12.08.519103 |
| Rights: |
info:eu-repo/semantics/OpenAccess |
| Accession Number: |
edsbas.87EAD298 |
| Database: |
BASE |