Optimized Secretome Sample Preparation From High Volume Cell Culture Media for LC-MS/MS Proteomic Analysis.

The cellular secretome is a rich source of biomarkers and extracellular signaling molecules, but proteomic profiling remains challenging, especially when processing culture volumes greater than 5 mL. Low protein abundance, high serum contamination, and sample loss during preparation limit reproducibility and sensitivity in mass spectrometry-based workflows. Here, we present an optimized and scalable protocol that integrates (i) 50 kDa molecular weight cutoff ultrafiltration, (ii) spin column depletion of abundant serum proteins, and (iii) acetone/TCA precipitation for protein recovery. This workflow enables balanced recovery of both low- and high-molecular-weight proteins while reducing background from serum albumin, thereby improving sensitivity, reproducibility, and dynamic range for LC-MS/MS analysis. Validated in human mesenchymal stromal cell cultures, the protocol is broadly applicable across diverse cell types and experimental designs, making it well-suited for biomarker discovery and extracellular proteomics. Key features • Enables efficient concentration and cleanup of ≥5-500 mL of conditioned media, suitable for low-abundance secreted protein recovery. • Combines 50 kDa ultrafiltration, optional HSA/IgG depletion, and acetone/TCA precipitation for robust removal of serum contaminants and improved signal-to-noise. • Adaptable to various mammalian cell types and serum-free or serum-containing media; scalable for adherent and suspension cultures.