Development of a phosphoproteomic workflow for the analysis of Chinese hamster ovary cell nuclei

Abstract

Phosphorylation of nuclear proteins has been widely recognized as a key chemical modification necessary for the control of mRNA transcription. Thus, the exact phosphorylation sites of nuclear proteins will be especially useful for revealing the molecular mechanisms that affect transcription in high producing cell lines and may be used to manipulate transcription rates. The challenge lies in the complexity of the nuclear proteome and in ensuring that upstream sample preparation is compatible with downstream analysis. In the current work the goal was to develop a solution based workflow to identify nuclear phosphoproteins with methods that are amenable to mass spectrometric analysis and to identify phosphorylated proteins in the nucleus of an industrially relevant cell line. By using proteomics approaches such as sample-clean up procedures, affinity chromatography and high sensitivity mass spectrometric based peptide sequencing, we have identified over 70 phosphorylation sites on 38 proteins in a CHO cell line.

Monoclonal antibodies are essential for the treatment of a wide variety of human diseases such as colon cancer and arthritis. Because of the complexity of monoclonal antibodies and the need for proper post-translational modifications (PTMs), mammalian host cells are used to create recombinant proteins that are fully functional in human targets. For more than twenty years, the leading host cells for protein therapeutics have been Chinese hamster ovary (CHO) cells because they are amenable to large scale culture conditions and are easily transfected with target DNA. Although CHO cells are widely used, the current selection process for high producing lines requires the testing of growth conditions for hundreds of cell lines in order to determine their productivity rates, which can be costly and time consuming. An alternative option for cell line selection that has not been fully explored is the screening of cell lines for protein markers such as transcriptional factors that are up- or down-regulated. This could potentially lead to more rapid selection criteria and increased production of recombinant proteins.