Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates
Background: Chinese hamster ovary (CHO) cells are the workhorse for obtaining recombinant proteins. Proteomic studies of these cells intend to understand cell biology and obtain more productive and robust cell lines for therapeutic protein production in the pharmaceutical industry. Because of the gr...
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| Format: | Article |
| Language: | English |
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Elsevier
2020-11-01
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| Series: | Electronic Journal of Biotechnology |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0717345820300452 |
| _version_ | 1818219211831377920 |
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| author | Saumel Pérez-Rodriguez Octavio T. Ramírez Mauricio A. Trujillo-Roldán Norma A. Valdez-Cruz |
| author_facet | Saumel Pérez-Rodriguez Octavio T. Ramírez Mauricio A. Trujillo-Roldán Norma A. Valdez-Cruz |
| author_sort | Saumel Pérez-Rodriguez |
| collection | DOAJ |
| description | Background: Chinese hamster ovary (CHO) cells are the workhorse for obtaining recombinant proteins. Proteomic studies of these cells intend to understand cell biology and obtain more productive and robust cell lines for therapeutic protein production in the pharmaceutical industry. Because of the great importance of precipitation methods for the processing of samples in proteomics, the acetone, methanol-chloroform (M/C), and trichloroacetic acid (TCA)-acetone protocols were compared for CHO cells in terms of protein recovery, band pattern resolution, and presence on SDS-PAGE. Results: Higher recovery and similar band profile with cellular homogenates were obtained using acetone precipitation with ultrasonic bath cycles (104.18 ± 2.67%) or NaOH addition (103.12 ± 5.74%), compared to the other two protocols tested. TCA-acetone precipitates were difficult to solubilize, which negatively influenced recovery percentage (77.91 ± 8.79%) and band presence. M/C with ultrasonic homogenization showed an intermediate recovery between the other two protocols (94.22 ± 4.86%) without affecting protein pattern on SDS-PAGE. These precipitation methods affected the recovery of low MW proteins (<15 kDa). Conclusions: These results help in the processing of samples of CHO cells for their proteomic study by means of an easily accessible, fast protocol, with an almost complete recovery of cellular proteins and the capture of the original complexity of the cellular composition. Acetone protocol could be incorporated to sample-preparation workflows in a straightforward manner and can probably be applied to other mammalian cell lines as well.How to cite: Pérez-Rodriguez S, Ramírez OT, Trujillo-Roldán MA et al. Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates. Electron J Biotechnol 2020;48. https://doi.org/10.1016/j.ejbt.2020.09.006. |
| first_indexed | 2024-12-12T07:36:03Z |
| format | Article |
| id | doaj.art-26e5750e988c4249a3220ea6bf1001c0 |
| institution | Directory Open Access Journal |
| issn | 0717-3458 |
| language | English |
| last_indexed | 2024-12-12T07:36:03Z |
| publishDate | 2020-11-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Electronic Journal of Biotechnology |
| spelling | doaj.art-26e5750e988c4249a3220ea6bf1001c02022-12-22T00:32:54ZengElsevierElectronic Journal of Biotechnology0717-34582020-11-01488694Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenatesSaumel Pérez-Rodriguez0Octavio T. Ramírez1Mauricio A. Trujillo-Roldán2Norma A. Valdez-Cruz3Programa de Investigación de Producción de Biomoléculas, Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Av Universidad 3000, Cd. Universitaria, Coyoacán, 04510 Ciudad de México, MexicoDepartamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Colonia Chamilpa, Cuernavaca 62210, Morelos, MexicoPrograma de Investigación de Producción de Biomoléculas, Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Av Universidad 3000, Cd. Universitaria, Coyoacán, 04510 Ciudad de México, MexicoPrograma de Investigación de Producción de Biomoléculas, Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Av Universidad 3000, Cd. Universitaria, Coyoacán, 04510 Ciudad de México, Mexico; Corresponding author.Background: Chinese hamster ovary (CHO) cells are the workhorse for obtaining recombinant proteins. Proteomic studies of these cells intend to understand cell biology and obtain more productive and robust cell lines for therapeutic protein production in the pharmaceutical industry. Because of the great importance of precipitation methods for the processing of samples in proteomics, the acetone, methanol-chloroform (M/C), and trichloroacetic acid (TCA)-acetone protocols were compared for CHO cells in terms of protein recovery, band pattern resolution, and presence on SDS-PAGE. Results: Higher recovery and similar band profile with cellular homogenates were obtained using acetone precipitation with ultrasonic bath cycles (104.18 ± 2.67%) or NaOH addition (103.12 ± 5.74%), compared to the other two protocols tested. TCA-acetone precipitates were difficult to solubilize, which negatively influenced recovery percentage (77.91 ± 8.79%) and band presence. M/C with ultrasonic homogenization showed an intermediate recovery between the other two protocols (94.22 ± 4.86%) without affecting protein pattern on SDS-PAGE. These precipitation methods affected the recovery of low MW proteins (<15 kDa). Conclusions: These results help in the processing of samples of CHO cells for their proteomic study by means of an easily accessible, fast protocol, with an almost complete recovery of cellular proteins and the capture of the original complexity of the cellular composition. Acetone protocol could be incorporated to sample-preparation workflows in a straightforward manner and can probably be applied to other mammalian cell lines as well.How to cite: Pérez-Rodriguez S, Ramírez OT, Trujillo-Roldán MA et al. Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates. Electron J Biotechnol 2020;48. https://doi.org/10.1016/j.ejbt.2020.09.006.http://www.sciencedirect.com/science/article/pii/S0717345820300452Cell homogenatesChinese hamster ovary cellsPrecipitation protocolsProtein precipitationProtein recoveryProtein solubilization |
| spellingShingle | Saumel Pérez-Rodriguez Octavio T. Ramírez Mauricio A. Trujillo-Roldán Norma A. Valdez-Cruz Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates Electronic Journal of Biotechnology Cell homogenates Chinese hamster ovary cells Precipitation protocols Protein precipitation Protein recovery Protein solubilization |
| title | Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates |
| title_full | Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates |
| title_fullStr | Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates |
| title_full_unstemmed | Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates |
| title_short | Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates |
| title_sort | comparison of protein precipitation methods for sample preparation prior to proteomic analysis of chinese hamster ovary cell homogenates |
| topic | Cell homogenates Chinese hamster ovary cells Precipitation protocols Protein precipitation Protein recovery Protein solubilization |
| url | http://www.sciencedirect.com/science/article/pii/S0717345820300452 |
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