LC-MS/MS Analysis to Study the Ubiquitin-Modified Proteome of Recombinant Chinese Hamster Ovary Cells

Ubiquitination is one of the most important post-translational modifications (PTMs) and involves the covalent attachment of ubiquitin to a lysine residue on a target protein. Despite ubiquitination playing a crucial role in regulating cellular processes, the ubiquitinated proteome has not been studi...

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Published inMethods in molecular biology (Clifton, N.J.) Vol. 2853; p. 191
Main Authors Selvaprakash, Karuppuchamy, Henry, Michael, Ryan, David, Meleady, Paula
Format Journal Article
LanguageEnglish
Published United States 2025
Subjects
Animals
CHO Cells
Chromatography, Liquid - methods
Cricetinae
Cricetulus
Immunoprecipitation - methods
Liquid Chromatography-Mass Spectrometry
Protein Processing, Post-Translational
Proteome
Proteomics - methods
Recombinant Proteins - genetics
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
Tandem Mass Spectrometry - methods
Ubiquitin - genetics
Ubiquitin - metabolism
Ubiquitination
Ubiquitin
Immunoprecipitation
diGly peptides
Chinese hamster ovary cells
Mass spectrometry
Proteomics
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Summary:Ubiquitination is one of the most important post-translational modifications (PTMs) and involves the covalent attachment of ubiquitin to a lysine residue on a target protein. Despite ubiquitination playing a crucial role in regulating cellular processes, the ubiquitinated proteome has not been studied extensively in recombinant Chinese hamster ovary (CHO) cells. Moreover, ubiquitination modification in CHO cells is likely to have an impact on protein function related to the efficient productivity of biopharmaceuticals. In this chapter, we describe a comprehensive protocol for ubiquitin di-Glycine (diGly) peptide enrichment using an immunoprecipitation method from recombinant CHO cell proteins followed by Liquid chromatography-Mass spectrometry (LC-MS) analysis of the ubiquitinated proteome. The methods described are also applicable to differential ubiquitinated proteomic studies.
ISSN:1940-6029
DOI:10.1007/978-1-0716-4104-0_13