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Using a peptide-based mass spectrometry approach to quantitate proteolysis of an intact heterogeneous procollagen substrate by BMP1 for antagonistic antibody screening

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Abstract

Proteases are critical proteins involved in cleaving substrates that may impact biological pathways, cellular processes, or disease progression. In the biopharmaceutical industry, modulating the levels of protease activity is an important strategy for mitigating many types of diseases. While a variety of analytical tools exist for characterizing substrate cleavages, in vitro functional screening for antibody inhibitors of protease activity using physiologically relevant intact protein substrates remains challenging. In addition, detecting such large protein substrates with high heterogeneity using high-throughput mass spectrometry screening has rarely been reported in the literature with concerns for assay robustness and sensitivity. In this study, we established a peptide-based in vitro functional screening assay for antibody inhibitors of mouse bone morphogenic protein 1 (mBMP1) metalloprotease using a heterogeneous recombinant 66-kDa mouse Procollagen I alpha 1 chain (mProcollagen) substrate. We compared several analytical tools including capillary gel electrophoresis Western blot (CE-Western blot), as well as both intact protein and peptide-based mass spectrometry (MS) to quantitate the mBMP1 proteolytic activity and its inhibition by antibodies using this heterogeneous mProcollagen substrate. We concluded that the peptide-based mass spectrometry screening assay was the most suitable approach in terms of throughput, sensitivity, and assay robustness. We then optimized our mBMP1 proteolysis reaction after characterizing the enzyme kinetics using the peptide-based MS assay. This assay resulted in Z′ values ranging from 0.6 to 0.8 from the screening campaign. Among over 1200 antibodies screened, IC50 characterization was performed on the top candidate hits, which showed partial or complete inhibitory activities against mBMP1.

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Acknowledgements

We thank Shirley Ng, Andy Chang, and Maureen Beresini for their assistance with automation; Eric Torres and Dan Hascall for their informatic support; and Suk Hyung, Sherry Ke Li, Yiming Xu, and Jonathan Josephs for reviewing the manuscript.

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Authors and Affiliations

  1. Department of Biochemical and Cellular Pharmacology, Genentech, South San Francisco, CA, USA

    Cong Wu, Ryan Cook, Elin Chee, William S. Sawyer, John C. Tran & Zhengmao Ye

  2. Department of Structural Biology, Genentech, South San Francisco, CA, USA

    Ping Wu

  3. University of Pennsylvania, Philadelphia, PA, USA

    Neha Srikumar

  4. Morphic Therapeutic, Waltham, MA, USA

    Hua Wang

  5. Department of Antibody Engineering, Genentech, South San Francisco, CA, USA

    Meredith Hazen & Isidro Hotzel

  6. Gilead Sciences, Foster City, CA, USA

    Elsa-Noah N’Diaye & Yichin Liu

  7. Department of Discovery Immunology, Genentech, South San Francisco, CA, USA

    Ning Ding

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  1. Cong Wu
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  2. Ryan Cook
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  3. Ping Wu
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  7. Hua Wang
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  11. Ning Ding
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Correspondence to Cong Wu.

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Wu, C., Cook, R., Wu, P. et al. Using a peptide-based mass spectrometry approach to quantitate proteolysis of an intact heterogeneous procollagen substrate by BMP1 for antagonistic antibody screening. Anal Bioanal Chem 414, 6601–6610 (2022). https://doi.org/10.1007/s00216-022-04220-2

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  • DOI: https://doi.org/10.1007/s00216-022-04220-2

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