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Phosphopeptide Enrichment and LC-MS/MS Analysis to Study the Phosphoproteome of Recombinant Chinese Hamster Ovary Cells

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Heterologous Protein Production in CHO Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1603))

Abstract

The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is one of the most important post-translational modifications that regulates many biological processes. The phosphoproteome has not been studied in any great detail in recombinant Chinese hamster ovary (CHO) cells to date despite phosphorylation playing a crucial role in regulating many molecular and cellular processes relevant to bioprocess phenotypes including, for example, transcription, translation, growth, apoptosis, and signal transduction. In this chapter, we provide a protocol for the phosphoproteomic analysis of Chinese hamster ovary cells using phosphopeptide enrichment with metal oxide affinity chromatography (MOAC) and immobilized metal affinity chromatography (IMAC) techniques, followed by site-specific identification of phosphorylated residues using LC-MS (MS2 and MS3) strategies.

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Acknowledgments

The authors acknowledge funding from Science Foundation Ireland (grant ref. 13/1A/1841) and the Horizon 2020 Marie Curie ITN programme – eCHO systems (grant ref.: 642663).

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Authors and Affiliations

  1. National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland

    Michael Henry, Orla Coleman,  Prashant, Martin Clynes & Paula Meleady

Authors
  1. Michael Henry
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  2. Orla Coleman
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  3. Prashant
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  4. Martin Clynes
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  5. Paula Meleady
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Corresponding author

Correspondence to Michael Henry .

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Editors and Affiliations

  1. National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland

    Paula Meleady

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Henry, M., Coleman, O., Prashant, Clynes, M., Meleady, P. (2017). Phosphopeptide Enrichment and LC-MS/MS Analysis to Study the Phosphoproteome of Recombinant Chinese Hamster Ovary Cells. In: Meleady, P. (eds) Heterologous Protein Production in CHO Cells. Methods in Molecular Biology, vol 1603. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6972-2_13

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  • DOI: https://doi.org/10.1007/978-1-4939-6972-2_13

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6971-5

  • Online ISBN: 978-1-4939-6972-2

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