Abstract
G-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins. Although implicated in almost all physiological processes in the human body, most of them remain unexploited, mostly because of the lack of specific ligands. The objective of this work is to develop a new mass-spectrometry-based technique capable of identifying new peptide ligands for GPCRs. The strategy is based on the incubation of cellular membranes overexpressing GPCRs with a mixture of peptides that contains potential ligands. Peptide ligands bind to the receptors, whereas other peptides remain in the binding buffer. Bound peptides are eluted from membranes and directly detected, identified, and characterized by MALDI TOF–TOF. The results reveal the efficacy of the procedure for selecting a specific ligand of GPCRs in both simple and complex mixtures of peptides. This new approach may offer direct purification, identification, and characterization of the new ligand in a single workflow. The proposed method is labeling-free and, unlike radio-binding and other techniques, it does not require a previously known labeled ligand of the studied GPCR. All these properties greatly reduce the experimental constraints. Moreover, because it is not based on the principle of a competitive specific binding, this technique constitutes a new tool to discover new ligands not only for known GPCRs, but also for orphan GPCRs.
Similar content being viewed by others
References
Perez DM (2003) The evolutionary triumphant G-protein-coupled receptor. Mol Pharmacol 63:1202–1205
Schlyer S, Horuk R (2006) I want a new drug: G-protein-coupled receptors in drug development. Drug Discov Today 11:481–493
Garland SL (2013) Are GPCRs still a source of new targets? J Biomol Screen 18:947–966
Robas N, O’Reilly M, Katugampola S, Fidock M (2003) Maximizing serendipity : strategies for identifying ligands for orphan G-protein-coupled receptors. Curr Opin 3:121–126
Zhang R, Xie X (2012) Tools for GPCR drug discovery. Acta Pharmacol Sin 33:372–384
Tang XL, Wang Y, Li DL, Luo J, Liu MY (2012) Orphan G protein-coupled receptors (GPCRs): biological functions and potential drug targets. Acta Pharmacol Sin 33:363–371
Chung S, Funakoshi T, Civelli O (2008) Orphan GPCR research. Br J Pharmacol 153:339–346
Thomsen W, Frazer J, Unett D (2005) Functional assays for screening GPCR targets. Curr Opin Biotechnol 16:655–665
Shemesh R, Toporik A, Levine Z, Hecht I, Rotman G, Wool A, Dahary D, Gofer E, Kliger Y, Soffer MA, Rosenberg A, Eshel D, Cohen Y (2008) Discovery and validation of novel peptide agonists for G-protein-coupled receptors. J Biol Chem 50:34643–34649
Lefkowitz RJ, Roth J, Pastan I (1970) Radio receptor assay of adrenocorticotropic hormone: new approach to assay of polypeptide hormones in plasma. Science 170:633–635
Navratilova I, Besnard J, Hopkins AL (2011) Screening for GPCR ligands using surface plasmon resonance. ACS Med Chem Lett 2:549–554
Jacoby E, Bouhelal R, Gerspacher M, Seuwen K (2006) The 7 TM G-protein-coupled receptor target family. ChemMedChem 1:761–782
Fang Y, Frutos GA, Verklereen R (2008) Label-free cell-based assays for GPCRs screening. Comb Chem High Throughput Screen 11:357–369
Whitehusrt CE, Annis DA (2008) Affinity selection-mass spectrometry and its emerging application to the high throughput screening of G protein-coupled-receptors. Comb Chem High Throughput Screen 11:427–438
Jonker N, Kool J, Irth H, Niessen WMA (2010) Recent developments in protein-ligand affinity mass spectrometry. Anal Bioanal Chem 399:2669–2681
Temporini C, Massolini G, Marucci G, Lambertucci C, Buccioni M, Volpini R, Calleri E (2013) Development of a new chromatographic tools based on A2A adenosine receptor subtype for ligand characterization and screening by FAC-MS. Anal Bioanal 405:837–845
Whitehusrt CE, Nazef N, Annis DA, Hou Y, Murphy DM, Spacciapoli P, Yao Z, Ziebell MR, Cheng CC, Shipps GW, Felsch JS, Lau D, Nash HM (2006) Discovery and characterization of orthosteric and allosteric muscarinic M2 acetylcholine receptor ligands by affinity selection-mass spectrometry. J Biomol Screen 11:194–207
Quinton L, Gilles N, de Pauw E (2009) TxXIIIA, an atypical homodimeric conotoxin found in the Conus textile venom. J Proteomics 72:219–226
Serradeil-Le Gal C, Lacour C, Valette G, Garcia G, Foulon L, Galindo G, Bankir L, Pouzet B, Guillon G, Barberis C, Chicot D, Jard S, Vilain P, Garcia C, Marty E, Raufaste D, Brossard G, Nisato D, Maffrand JP, Le Fur G (1996) Characterization of SR 121463A, a highly potent and selective, orally active vasopressin V2 receptor antagonist. J Clin Invest 98:2729–2738
Vauquelin G, Von Mentzer B (2007) Radioligand binding studies. In: Vauquelin G, von Mentzer P (eds) G protein-coupled receptors: molecular pharmacology. Wiley & Sons Ltd, Chichester, pp 29–52
Favreau P, Menin L, Michhaleet S, Perret F, Cheneval O, Stöcklin M, Bulet P, Stöcklin R (2006) Mass spectrometry strategies for venom mapping and peptide sequencing from crude venoms: case applications with single arthropod specimen. Toxicon 47:676–687
O'Connel T, Ramsay J, Rieth SF, Shapiro MJ, Stroh JG (2014) Solution-based indirect affinity selection mass spectrometry- a general tool for high throughput screening of pharmaceutical compound libraries. Anal Chem 86:7413–7420
Acknowledgements
This project was financially supported by the University of Liège (Special funding for the Research 2011-2012) and the Brazilian research agency CNPq - National Council of Scientific and Technologic Development (process number: 246334/2012-6).
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
(PDF 1.32 MB)
Rights and permissions
About this article
Cite this article
Cologna, C.T., Gilles, N., Echterbille, J. et al. Mass-spectrometry-based method for screening of new peptide ligands for G-protein-coupled receptors. Anal Bioanal Chem 407, 5299–5307 (2015). https://doi.org/10.1007/s00216-015-8692-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-015-8692-4