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Improved Immunoprecipitation to Mass Spectrometry Method for the Enrichment of Low-Abundant Protein Targets

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Proteomic Profiling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2261))

Abstract

Immunoprecipitation (IP) is commonly used upstream of mass spectrometry (MS) as an enrichment tool for low-abundant protein targets. However, several aspects of the classical IP procedure such as nonspecific protein binding to the isolation matrix, detergents or high salt concentrations in wash and elution buffers, and antibody chain contamination in elution fractions render it incompatible with downstream mass spectrometry analysis. Here, we discuss an improved IP-MS workflow that is designed to minimize sample prep time and these contaminants. The method employs biotinylated antibodies to the targets of interest and streptavidin magnetic beads that exhibit low background binding. In addition, alterations in the elution protocol and subsequent MS sample prep were made to reduce time and antibody leaching in the eluent, minimizing potential ion suppression effects and thereby maximizing detection of multiple target antigens and interacting proteins.

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Authors and Affiliations

  1. Thermo Fisher Scientific, Rockford, IL, USA

    Penny Jensen, Bhavin Patel, Suzanne Smith & Barbara Kaboord

  2. Nisarga Biotech Pvt. Ltd., Satara, Maharashtra, India

    Renuka Sabnis

Authors
  1. Penny Jensen
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  2. Bhavin Patel
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  3. Suzanne Smith
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  4. Renuka Sabnis
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  5. Barbara Kaboord
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Corresponding author

Correspondence to Barbara Kaboord .

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Editors and Affiliations

  1. Bio-Rad Laboratories GmbH, Feldkirchen, Germany

    Anton Posch

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Jensen, P., Patel, B., Smith, S., Sabnis, R., Kaboord, B. (2021). Improved Immunoprecipitation to Mass Spectrometry Method for the Enrichment of Low-Abundant Protein Targets. In: Posch, A. (eds) Proteomic Profiling. Methods in Molecular Biology, vol 2261. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1186-9_14

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  • DOI: https://doi.org/10.1007/978-1-0716-1186-9_14

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1185-2

  • Online ISBN: 978-1-0716-1186-9

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