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.
Keywords: Chinese hamster ovary cells; Phosphopeptide enrichment; Phosphoproteomics; Site specific phosphorylation.