Secretome Processing for Proteomics: A Methods Comparison

The cancer cell secretome comprises a treasure-trove for biomarkers since it reflects cross-talk between tumor cells and their surrounding environment. In this study, we evaluated six secretome sample processing workflows coupled to single-shot mass spectrometry: 1. Protein concentration by ultrafiltration with a molecular weight cut-off (MWCO) filter and sample preparation through in-gel digestion (IGD); 2. Acetone protein precipitation coupled to IGD; 3. MWCO filter-based protein concentration followed by to in-solution digestion (ISD); 4. Acetone protein precipitation coupled to ISD; 5. Direct ISD; 6. Secretome lyophilization and ISD. To this end, we assessed workflow triplicates in terms of total number of protein identifications, unique identifications, reproducibility of protein identification and quantification and detectability of small proteins with important functions in cancer biology such as cytokines, chemokines and growth factors.