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

Paré B, Deschênes LT, Pouliot R, Dupré N, Gros-Louis F - Front Cell Neurosci (2016)

Bottom Line: To facilitate ongoing research involving secreted proteins, we revisited cell culture protocols and whole secreted protein enrichment protocols.The well-established carrier-based TCA-DOC protein precipitation method was consistently found to give higher protein recovery yield.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Regenerative Medicine, Laval University Experimental Organogenesis Research Center/LOEX, CHU de Québec Research Center - Enfant-Jésus HospitalQuébec, QC, Canada; Department of Surgery, Faculty of Medicine, Laval UniversityQuébec, QC, Canada.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Visualization of whole secreted protein and quantification of the protein spots resolved with 2D-DIGE at the optimal induction condition. Conjugation of each CyDye, Cy2 (A), Cy3 (B), and Cy5 (C), to precipitated proteins extracted from fibroblast conditioned-medium via the TCA-DOC method and resolved by 2D-DIGE is shown. Please note that all technical replicates (disease and control) obtained for the 48 h post-confluence induction condition were pooled together prior CyDye conjugation and loaded on gel. (D) Represents a fusion image of the three different CyDyes. Each individual spot is circled. The number of detected spots (502) is indicated at the far right. Equal number of spots is detected for each of the tested CyDyes indicating that each of the specific tested CyDyes bond with the same effectiveness and therefore that the conjugation efficiency is independent of the CyDye fluors.
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Figure 3: Visualization of whole secreted protein and quantification of the protein spots resolved with 2D-DIGE at the optimal induction condition. Conjugation of each CyDye, Cy2 (A), Cy3 (B), and Cy5 (C), to precipitated proteins extracted from fibroblast conditioned-medium via the TCA-DOC method and resolved by 2D-DIGE is shown. Please note that all technical replicates (disease and control) obtained for the 48 h post-confluence induction condition were pooled together prior CyDye conjugation and loaded on gel. (D) Represents a fusion image of the three different CyDyes. Each individual spot is circled. The number of detected spots (502) is indicated at the far right. Equal number of spots is detected for each of the tested CyDyes indicating that each of the specific tested CyDyes bond with the same effectiveness and therefore that the conjugation efficiency is independent of the CyDye fluors.

Mentions: A pool of precipitated proteins collected from all the samples tested in this study, using the TCA-DOC method 48 h post-confluence and induction, were conjugated with each CyDyes (2, 3, and 5) and resolved with 2D-DIGE. The results show that each CyDyes bind at the same effectiveness to the proteins, giving an equal number of detected spots (502) for each of the conjugation (Figure 3) indicating that the choice of a specific CyDye does not affect or interfere with protein detection. Conjugation of the samples for each tested conditions was done in a randomized fashion between the two CyDyes used in the study (Cy3 and Cy5). The conjugated proteins mixture, precipitated from fibroblast conditioned-medium for two different conditions, was subjected to isoelectric focusing followed by a 10–18% SDS-PAGE gradient migration in the 2nd dimension. To determine the number of spots detected, the Delta 2D analysis software V4.2 (Decodon, Greifswald, Germany) was used. A data pool, comprised of all the gels and the CyDyes, was created. Quantification of the number of spots detected between different induction conditions and precipitation methods (TCA-DOC post-confluence, 24 h of incubation/TCA-DOC, post-confluence, 48 h of incubation/TCA-NLS-THF, post-confluence, 48 h of incubation) was also assessed. In line with our previous results showing that the TCA-DOC 48 h post-confluency was given better protein recovery yield, more spots were also detected using this method when compared to the two other conditions tested (Figure 4).


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

Paré B, Deschênes LT, Pouliot R, Dupré N, Gros-Louis F - Front Cell Neurosci (2016)

Visualization of whole secreted protein and quantification of the protein spots resolved with 2D-DIGE at the optimal induction condition. Conjugation of each CyDye, Cy2 (A), Cy3 (B), and Cy5 (C), to precipitated proteins extracted from fibroblast conditioned-medium via the TCA-DOC method and resolved by 2D-DIGE is shown. Please note that all technical replicates (disease and control) obtained for the 48 h post-confluence induction condition were pooled together prior CyDye conjugation and loaded on gel. (D) Represents a fusion image of the three different CyDyes. Each individual spot is circled. The number of detected spots (502) is indicated at the far right. Equal number of spots is detected for each of the tested CyDyes indicating that each of the specific tested CyDyes bond with the same effectiveness and therefore that the conjugation efficiency is independent of the CyDye fluors.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814560&req=5

Figure 3: Visualization of whole secreted protein and quantification of the protein spots resolved with 2D-DIGE at the optimal induction condition. Conjugation of each CyDye, Cy2 (A), Cy3 (B), and Cy5 (C), to precipitated proteins extracted from fibroblast conditioned-medium via the TCA-DOC method and resolved by 2D-DIGE is shown. Please note that all technical replicates (disease and control) obtained for the 48 h post-confluence induction condition were pooled together prior CyDye conjugation and loaded on gel. (D) Represents a fusion image of the three different CyDyes. Each individual spot is circled. The number of detected spots (502) is indicated at the far right. Equal number of spots is detected for each of the tested CyDyes indicating that each of the specific tested CyDyes bond with the same effectiveness and therefore that the conjugation efficiency is independent of the CyDye fluors.
Mentions: A pool of precipitated proteins collected from all the samples tested in this study, using the TCA-DOC method 48 h post-confluence and induction, were conjugated with each CyDyes (2, 3, and 5) and resolved with 2D-DIGE. The results show that each CyDyes bind at the same effectiveness to the proteins, giving an equal number of detected spots (502) for each of the conjugation (Figure 3) indicating that the choice of a specific CyDye does not affect or interfere with protein detection. Conjugation of the samples for each tested conditions was done in a randomized fashion between the two CyDyes used in the study (Cy3 and Cy5). The conjugated proteins mixture, precipitated from fibroblast conditioned-medium for two different conditions, was subjected to isoelectric focusing followed by a 10–18% SDS-PAGE gradient migration in the 2nd dimension. To determine the number of spots detected, the Delta 2D analysis software V4.2 (Decodon, Greifswald, Germany) was used. A data pool, comprised of all the gels and the CyDyes, was created. Quantification of the number of spots detected between different induction conditions and precipitation methods (TCA-DOC post-confluence, 24 h of incubation/TCA-DOC, post-confluence, 48 h of incubation/TCA-NLS-THF, post-confluence, 48 h of incubation) was also assessed. In line with our previous results showing that the TCA-DOC 48 h post-confluency was given better protein recovery yield, more spots were also detected using this method when compared to the two other conditions tested (Figure 4).

Bottom Line: To facilitate ongoing research involving secreted proteins, we revisited cell culture protocols and whole secreted protein enrichment protocols.The well-established carrier-based TCA-DOC protein precipitation method was consistently found to give higher protein recovery yield.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Regenerative Medicine, Laval University Experimental Organogenesis Research Center/LOEX, CHU de Québec Research Center - Enfant-Jésus HospitalQuébec, QC, Canada; Department of Surgery, Faculty of Medicine, Laval UniversityQuébec, QC, Canada.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus