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SPRi-MALDI MS: characterization and identification of a kinase from cell lysate by specific interaction with different designed ankyrin repeat proteins

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Abstract

We report on the direct coupling of surface plasmon resonance imaging (SPRi) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) for the investigation of specific, non-covalent interactions, using the example of designed ankyrin repeat proteins (DARPins) and ribosomal protein S6 kinase 2 (RPS6KA2) directly from lysate of SH-SY5Y cells, derived from human bone marrow. Due to an array format, tracing of binding kinetics of numerous DARPins simultaneously and in real time becomes possible. By optimizing both the proteolytic digest directly on the SPRi chip (amount of trypsin, incubation time, and temperature) as well as the MALDI matrix application (concentration of matrix and number of spray cycles), we are able to identify the specific interaction with RPS6KA2 directly from the cell lysate at a surface coverage of only 0.8 fmol/mm2.

Workflow of the direct coupling of SPRi with MALDI mass spectrometry

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Acknowledgements

We thank Horiba Jobin Yvon for the loan of an SPRi instrument and Bruker Daltonics and Horiba Jobin Yvon for jointly funding 1.5 years of a Ph.D. studentship awarded to U.A. We also thank all current and former members of the High-Throughput Binder Selection Facility for their contribution to the establishment of the semi-automated ribosome display that resulted in the used DARPin binders. The RPS6KA2 target used for DARPin selection and the experiments described here was kindly provided by Dr. Susanne Müller-Knapp and Tracy Keates (SGC Oxford). Chia-Lung Yang (group of Prof. Olga Shakhova) kindly provided the cell lysate of SH-SY5Y (Department of Oncology, University Hospital Zurich).

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Authors and Affiliations

  1. Department of Chemistry and Applied Biosciences, ETH Zurich, Vladimir-Prelog-Weg 3, 8093, Zurich, Switzerland

    Ulrike Anders & Renato Zenobi

  2. Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland

    Jonas V. Schaefer & Andreas Plückthun

  3. Horiba Jobin Yvon S.A.S., Avenue de la Vauve-Passage Jobin Yvon, CS 45002, 91120, Palaiseau, France

    Fatima-Ezzahra Hibti & Chiraz Frydman

  4. Bruker Daltonics, Fahrenheitstr. 4, 28359, Bremen, Germany

    Detlev Suckau

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  1. Ulrike Anders
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Anders, U., Schaefer, J.V., Hibti, FE. et al. SPRi-MALDI MS: characterization and identification of a kinase from cell lysate by specific interaction with different designed ankyrin repeat proteins. Anal Bioanal Chem 409, 1827–1836 (2017). https://doi.org/10.1007/s00216-016-0127-3

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