Antigen Identification for Cell-Binding Antibodies Using Ligand-Directed Crosslinking and Biotin Transfer
Panning approaches using antibody libraries often result in the isolation of antibodies that bind to cells through an unknown cellular receptor. Here, we describe a protocol that uses ligand-directed crosslinking with the aminooxy-sulfhydryl-biotin (ASB) trifunctional crosslinker followed by a proteomic analysis to identify the cellular receptors for orphan ligands. We describe the synthesis of the ASB crosslinker, labelling of the ligand with ASB, and cell binding of the labelled ligands. Next, biotin affinity purification and trypsin digestion of cell surface proteins that have been crosslinked by ASB are described. Lastly, several hints and tips to improve the proteomic analysis for these types of samples are provided.
Key wordsReceptor identification Crosslinking Proteomics Mass spectrometry Antibodies
We would like to thank the National Research Council Canada for supporting this research.
- 4.Xiao X, Chen Y, Mugabe S, Gao C, Tkaczyk C, Mazor Y, Pavlik P, Wu H, Dall'Acqua W, Chowdhury PS (2015) A novel dual expression platform for high throughput functional screening of phage libraries in product like format. PLoS One 10(10):e0140691. https://doi.org/10.1371/journal.pone.0140691CrossRefPubMedPubMedCentralGoogle Scholar
- 6.Frei AP, Jeon OY, Kilcher S, Moest H, Henning LM, Jost C, Pluckthun A, Mercer J, Aebersold R, Carreira EM, Wollscheid B (2012) Direct identification of ligand-receptor interactions on living cells and tissues. Nat Biotechnol 30(10):997–1001. https://doi.org/10.1038/nbt.2354CrossRefPubMedGoogle Scholar
- 8.Tremblay TL, Hill JJ (2017) Biotin-transfer from a trifunctional crosslinker for identification of cell surface receptors of soluble protein ligands. Sci Rep 7:46574. https://doi.org/10.1038/srep46574
- 12.Palagi PM, Walther D, Quadroni M, Catherinet S, Burgess J, Zimmermann-Ivol CG, Sanchez JC, Binz PA, Hochstrasser DF, Appel RD (2005) MSight: an image analysis software for liquid chromatography-mass spectrometry. Proteomics 5(9):2381–2384. https://doi.org/10.1002/pmic.200401244CrossRefGoogle Scholar
- 13.Sobotzki N, Schafroth MA, Rudnicka A, Koetemann A, Marty F, Goetze S, Yamauchi Y, Carreira EM, Wollscheid B (2018) HATRIC-based identification of receptors for orphan ligands. Nat Commun 9 (1): 1519. https://doi.org/10.1038/s41467-018-03936-z