Comparative analysis of differentially secreted proteins in serum-free and serum-containing media by using BONCAT and pulsed SILAC

Abstract

Despite the increased interest in secretomes associated with paracrine/autocrine mechanisms, the majority of mass spectrometric cell secretome studies have been performed using serum-free medium (SFM). On the other hand, serum-containing medium (SCM) is not recommended very much because the secretome obtained with SCM is easily contaminated with fetal bovine serum (FBS) proteins. In this study, through the combination of bioorthogonal non-canonical amino acid tagging (BONCAT) and pulsed-SILAC (pSILAC), we analyzed differentially secreted proteins between SFM and SCM in a cancer-derived human cell, U87MG, and a mesenchymal stem cell derived from human Wharton’s jelly (hWJ-MSCs). In most cases, the bioinformatic tools predicted a protein to be truly secretory when the secretion level of the protein was more in SCM than in SFM. In the case of hWJ-MSCs, the amount of proteins secreted in SCM for 24 hours was larger than that of SFM (log 2 fold change = 0.96), even considering different cell proliferation rates. hWJ-MSCs proteins secreted more in SCM included several positive markers of MSC paracrine factors implicated in angiogenesis, neurogenesis and osteogenesis, and upstream regulators of cell proliferation. Our study suggests the analysis of the secretome should be processed in SCM that promotes cell proliferation and secretion.

Secretomics to Discover Regulators in Diseases

“…These studies have provided interesting results contributing to new insights and research directions in diverse fields. Cell culture is a relatively easy method for harvesting secretomes by discriminating expected contaminants such as serum-originated proteins, and for mimicking pathophysiological conditions such as hypoxia, diabetes, and anti-cancer drug treatment [47,48,49]. After culturing cells to appropriate confluence, incubation using serum-free medium is essential to harvest the secretome so as to avoid contamination of serum components [47,48].…”

“…Cell culture is a relatively easy method for harvesting secretomes by discriminating expected contaminants such as serum-originated proteins, and for mimicking pathophysiological conditions such as hypoxia, diabetes, and anti-cancer drug treatment [47,48,49]. After culturing cells to appropriate confluence, incubation using serum-free medium is essential to harvest the secretome so as to avoid contamination of serum components [47,48]. However, it has been proposed that a serum-free medium might further contaminate the cell secretory protein profile owing to poor cell viability, thereby reducing the level of capture of the true physiological secretome [47,50].…”

“…After culturing cells to appropriate confluence, incubation using serum-free medium is essential to harvest the secretome so as to avoid contamination of serum components [47,48]. However, it has been proposed that a serum-free medium might further contaminate the cell secretory protein profile owing to poor cell viability, thereby reducing the level of capture of the true physiological secretome [47,50]. Depending on the cell type studied, the length of incubation in serum-free medium can vary, which might dramatically influence the secretome profiles [51].…”

“…One of the major challenges of secretomics is the inherent sensitivity of the mass spectrometer to cover the wide dynamic range for the low abundance of genuine secreted proteins over the high abundant proteins derived from the serum, which is essential for standard maintenance of the sample [148]. High-resolution mass spectrometers such as Q-Exactive HF, in combination with the optimized sample preparation schemes to enrich newly synthesized secreted proteins facilitate identification of low-level secreted protein from the optimized CM for cell growth [47,149,150,151,152]. Advances in the sensitivity of secretome analysis have enabled identifying samples that are highly susceptible to the media condition such as stem cells [153] and primary cells [154], as well as to compare the secreted protein profiles between serum-free and serum-containing CM [47].…”

Fishing for newly synthesized proteins with phosphonate-handles

“…streptavidin) devised to specifically purify these co-translationally tagged POIs 16 . The added flexibility to reverse affinity handleprobe pairs 14 and compatibility with pulsed-SILAC 17,18 have drastically expanded the utility of BONCAT to secretomics 19 , degradomics 20 , and many other innovative applications [21][22][23][24] .…”