An Optimized Approach to Recover Secreted Proteins from Fibroblast Conditioned-Media for Secretomic Analysis

The proteins secreted by a particular type of cell, the secretome, play important roles in the regulation of many physiological processes via paracrine/autocrine mechanisms, and they are of increasing interest to help understanding rare diseases and to identify potential biomarkers and therapeutic targets. To facilitate ongoing research involving secreted proteins, we revisited cell culture protocols and whole secreted protein enrichment protocols. A reliable method for culturing and precipitating secreted protein from patient-derived fibroblast conditioned-medium was established. The method is based on the optimization of cell confluency and incubation time conditions. The well-established carrier-based TCA-DOC protein precipitation method was consistently found to give higher protein recovery yield. According to our results, we therefore propose that protein enrichment should be performed by TCA-DOC precipitation method after 48 h at 95% of confluence in a serum-deprived culture medium. Given the importance of secreted proteins as a source to elucidate the pathogenesis of rare diseases, especially neurological disorders, this approach may help to discover novel candidate biomarkers with potential clinical significance.


• Fibroblast cells extraction
o Incubate the biopsies in 0.05% thermolysin overnight at 4°C. o Separate the epidermis from the dermis mechanically. o Isolate fibroblasts from the dermis after treatment with 0,2 IU/ml collagenase H.
• Fibroblasts induction o Wash 3 times with 10 ml of phosphate-buffered saline (PBS). o Deprive fibroblasts of serum (DMEM supplemented with 100 IU/ml penicillin G and 25 µg/ml gentamicin). o Incubate for the desired time (24h or 48h) in 5% CO 2 at 37°C. o Collect the supernatants and centrifuge at 300g for 10 minutes (4°C). Keep at -80°C until use.
• TCA-DOC precipitation o Thaw on ice the supernatants in high-speed centrifuge tube. o Add 1% (v/v) of a 2% sodium deoxycholate solution to the supernatants and incubate on ice for 30 minutes after mixing. o Add 100% trichloroacetic acid to a final concentration of 7,5% (v/v) and incubate on ice for 60 minutes after mixing. o Precipitate the proteins by centrifugation (15,000g for 20 minutes at 4°C) and discard the supernatants. o Add 20 ml of 100% ice-cold (-20°C) acetone to the pellets, vortex gently and keep at -20°C for 5 minutes. o Centrifuge (15,000g for 5 minutes at 4°C) and discard the supernatants. o Add 5 ml of 100% ice-cold (-20°C) acetone, vortex gently and keep at -20°C for 5 minutes. o Centrifuge (15,000g for 5 minutes at 4°C) and discard the supernatants. o Air-dry the pellets in a chemical hood for 30 minutes and dissolve in 210 µl of Isoelectric Focusing (IEF) buffer, pH 8.5. o Vortex the samples, centrifuge at 15,000g for 10 minutes at room temperature and keep at -80°C until use.
• TCA-NLS-THF precipitation o Thaw on ice the supernatants in high-speed centrifuge tube. o Add 1% (v/v) of a 2% sodium layroyl sarcosine solution to the supernatants and incubate on ice for 30 minutes after mixing. o Add 100% trichloroacetic acid to a final concentration of 7,5% (v/v) and incubate on ice for 60 minutes after mixing.  (Table 2). o Centrifuge (21,000g for 30 seconds at room temperature) and collect the supernatant. § First dimension electrophoresis • Rehydrate the Immobiline 230 DryStrip pH 3-11 NL, 24 cm at room temperature overnight in 450 µl of a mix of the remaining reduced supernatant and rehydration solution (Table 2). • Migrate the strips following the protocol mentioned in table 3.
• Recover the strips with Plus One DryStrip cover fluid during migration and keep them at -80°C until use. § Second dimension electrophoresis • Cast 10-18% gradient polyacrylamide gels (26 X 20 cm) with the DALTsix gel caster and the DALTsix gradient maker. • Cast the gels (light then heavy gel) then the moving solution following the specific recipe (Table 2).