Identification of secreted proteins by comparison of protein abundance in conditioned media and cell lysates

Analysis of the full spectrum of secreted proteins in cell culture is complicated by leakage of intracellular proteins from damaged cells. To address this issue, we compared the abundance of individual proteins between the cell lysate and the conditioned medium, reasoning that secreted proteins should be relatively more abundant in the conditioned medium. Marked enrichment for signal-peptide-bearing proteins with increasing conditioned media to cell lysate ratio, as well loss of this signal following brefeldin A treatment, confirmed the sensitivity and specificity of this approach. The subset of proteins demonstrating increased conditioned media to cell lysate ratio in the presence of Brefeldin A identified candidates for unconventional secretion via a pathway independent of ER to Golgi trafficking.