In-Depth Proteomic Quantification of Cell Secretome in Serum-Containing Conditioned Medium

Secreted proteins play key roles
during cellular communication,
proliferation, and migration. The comprehensive profiling of secreted
proteins in serum-containing culture media is technically challenging.
Most studies have been performed under serum-free conditions. However,
these conditions might alter the status of the cells. Herein, we describe
an efficient strategy that avoids the disturbance of serum by combining
metabolic labeling, protein “equalization,” protein
fractionation, and filter-aided sample preparation, called MLEFF,
enabling the identification of 534 secreted proteins from HeLa conditioned
media, including 31 cytokines, and growth factors. This MLEFF strategy
was also successfully applied during a comparative secretome analysis
of two human hepatocellular carcinoma cell lines with differentially
metastatic potentials, enabling the quantification of 61 significantly
changed proteins involved in tumor invasion and metastasis.