Profiling of the secretome of human cancer cells: Preparation of supernatant for proteomic analysis

Abstract

Secretomic analysis requires removal of serum proteins from cell-culture media. We evaluate the proteins washed from cells prepared in bovine serum-supplemented medium. PBS and serum-free-medium (SFM) were the washing solutions. A Bradford assay was used for total protein concentration and a 1D gel and LC-MS/MS, to assign the protein to human or bovine origin. For both wash solutions, all bovine protein had been removed by the third wash, without compromising the number of living cells. Further washes reduced the number of living cells, especially when using PBS. Proteomic analysis of wash supernatant showed that SFM induced greater lysis of dead cells. Three washes were sufficient to minimize the effects on cell viability, while still removing serum proteins. Washing in SFM resulted in contamination of the wash supernatant with lysed dead cell proteins. Washed cells were incubated in SFM and exposed to ionizing radiation. Analysis of the supernatant showed an increase in human cytoplasmic, plasma membrane, and nuclear protein following irradiation. Secreted proteins were also detected, but in smaller quantities. The significance of these findings extend to in vitro studies of bystander phenomena, since the proteins of lysed dead cells may participate in driving bystander responses.

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

“…This technique has been previously shown to induce secretion of proteins (Villarreal et al, 2013 ). Using this approach, previous work also demonstrated that short serum deprivation periods did not affect cellular viability from 48 h and inward (Del Galdo et al, 2010 ; Villarreal et al, 2013 ; Rogers et al, 2014 ). Fibroblast's supernatants were therefore collected at different confluence levels and at different collection times after serum deprivation; 24 h pre-confluence, 48 h pre-confluence, 24 h post-confluence, and 48-post confluence (please note that the term pre-confluence is defined as a confluence of 40% and post-confluence as a confluence of 95%).…”

“…The maximum recovery yield was obtained when collecting the conditioned supernatants 48 h after induction when fibroblasts were at 95% of confluence with the TCA-DOC precipitation method. Washing the cells three times with PBS before serum deprivation ruled out possible contaminants present in the bovine serum (Rogers et al, 2014 ). It cannot be excluded however that some intracellular proteins, released in the medium by cellular lysis, might be present in the collected supernatants.…”

“…Nonetheless, since the serum-deprived protocol used to extract proteins was the same for all tested conditions, it is unlikely that the presence of lysis-derived contaminants would interfere with the analysis and give false-positive results. Moreover, it has been previously shown that the removal of bovine protein, especially when using PBS, seems not to compromise the number of living cells and to minimize the effects on cell lysis and viability (Rogers et al, 2014 ). A comparative approach between the conditioned-medium and cytosolic extract should however be performed in order to normalize the results for the presence of minimized lysis-derived contaminants.…”

Variations of secretome profiles according to conditioned medium preparation: The example of human mesenchymal stem cell‐derived adipocytes

“…Although it is known that serum deprivation can deeply affect the cellular state, in our study, according to mRNA expression levels, cells did not seem to suffer and presented a phenotype similar to that of adipocyte control. In a recent study, the impact of the washing buffers and of the number of washes were evaluated in a secretome analysis of a human cancer cell line . An important observation of this work was that washing in serum‐free medium resulted in greater lysis of the cells than washing in PBS, and that more than three washes reduced the number of viable cells.…”

Effect of Recombinant Human Erythroferrone Protein on Hepcidin Gene (Hamp1) Expression in HepG2 and HuH7 Cells

“…Each 10 μL of standard BSA (0.0625, 0.125, 0.25, 0.5, 1, 1.5 and 2 mg/mL) or sample solution was mixed with 1 mL of working Bradford reagent (5× dilution of the stock Bradford solution). The mixtures were mixed well and incubated in dim light at room temperature for 10 min and the optical density at 595 nm was determined [ 17 ].…”