Identifying the CHO secretome using mucin-type O-linked glycosylation and click-chemistry

Chinese hamster ovary cells (CHO) are the most common cell line used in the production of therapeutic proteins. Understanding the complex pattern of secreted host cell proteins (HCP) that are released by CHO cells will facilitate the development of new recombinant protein production processes. In this study, we have adapted the N-azido-galactosamine (GalNAz) metabolic labeling method to enable the mass spectrometry identification and quantification of secreted proteins in cell culture media. CHO DG44 and CHO-S cells were cultured in media containing GalNAz, which was metabolically incorporated into mucin-type O-linked glycans of secreted proteins. These proteins were effectively enriched using click-chemistry from the cell culture media, allowing for the analysis of secreted proteins across multiple days of cell growth. When compared to the standard method for secretome analysis, the GalNAz method not only increased the total number of proteins identified but dramatically improved the quality of data by decreasing the number of background proteins (cytosolic or nuclear) to essentially zero.