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Antigen Identification for Cell-Binding Antibodies Using Ligand-Directed Crosslinking and Biotin Transfer

  • Tammy-Lynn Tremblay
  • Jennifer J. HillEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2024)

Abstract

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 words

Receptor identification Crosslinking Proteomics Mass spectrometry Antibodies 

Notes

Acknowledgments

We would like to thank the National Research Council Canada for supporting this research.

References

  1. 1.
    Zhao A, Tohidkia MR, Siegel DL, Coukos G, Omidi Y (2016) Phage antibody display libraries: a powerful antibody discovery platform for immunotherapy. Crit Rev Biotechnol 36(2):276–289.  https://doi.org/10.3109/07388551.2014.958978CrossRefPubMedGoogle Scholar
  2. 2.
    Roodink I, Franssen M, Zuidscherwoude M, Verrijp K, van der Donk T, Raats J, Leenders WP (2010) Isolation of targeting nanobodies against co-opted tumor vasculature. Lab Investig 90(1):61–67.  https://doi.org/10.1038/labinvest.2009.107CrossRefPubMedGoogle Scholar
  3. 3.
    Stutz CC, Zhang X, Shusta EV (2014) Combinatorial approaches for the identification of brain drug delivery targets. Curr Pharm Des 20(10):1564–1576CrossRefGoogle Scholar
  4. 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
  5. 5.
    Ten Haaf A, Pscherer S, Fries K, Barth S, Gattenlohner S, Tur MK (2015) Phage display-based on-slide selection of tumor-specific antibodies on formalin-fixed paraffin-embedded human tissue biopsies. Immunol Lett 166(2):65–78.  https://doi.org/10.1016/j.imlet.2015.05.013CrossRefPubMedGoogle Scholar
  6. 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
  7. 7.
    Frei AP, Moest H, Novy K, Wollscheid B (2013) Ligand-based receptor identification on living cells and tissues using TRICEPS. Nat Protoc 8(7):1321–1336.  https://doi.org/10.1038/nprot.2013.072CrossRefPubMedGoogle Scholar
  8. 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
  9. 9.
    Tyanova S, Temu T, Cox J (2016) The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat Protoc 11(12):2301–2319.  https://doi.org/10.1038/nprot.2016.136CrossRefPubMedGoogle Scholar
  10. 10.
    Carvalho PC, Lima DB, Leprevost FV, Santos MD, Fischer JS, Aquino PF, Moresco JJ, Yates JR 3rd, Barbosa VC (2016) Integrated analysis of shotgun proteomic data with PatternLab for proteomics 4.0. Nat Protoc 11(1):102–117.  https://doi.org/10.1038/nprot.2015.133CrossRefPubMedGoogle Scholar
  11. 11.
    Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82(1):70–77CrossRefGoogle Scholar
  12. 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. 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

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Human Health TherapeuticsNational Research Council CanadaOttawaCanada

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