Improving Proteome Coverage for Hs578T Breast Cancer Cell-Line due to Efficient Interfering Removal

Document Type : Original Article (s)

Authors

1 Department of Medical Biotechnology, School of Allied Medical Sciences, Iran University of Medical Sciences, Tehran, Iran

2 Assistant Professor; Department of Medical Biotechnology, School of Allied Medical Sciences, Iran University of Medical Sciences, Tehran, Iran

3 MSc Student, Student Research Committee, Department of Medical Biotechnology, School of Allied Medical Sciences, Iran University of Medical Sciences, Tehran, Iran

4 Professor, Department of Medical Biotechnology, School of Allied Medical Sciences, Iran University of Medical Sciences, Tehran, Iran

5 Associate Professor, Immunology Research Center, Iran University of Medical Sciences, Tehran, Iran

Abstract

Background: In spite of the wide use, low proteome coverage and fuzzy patterns are the most important deterrents for the clinical applications of gel-based proteomic studies. So herein, we tried to increase the 2-dimentional proteome coverage of Hs578T breast cancer cells via investigating the efficacy of the three common techniques, usually used for interfering removal.Methods: Hs578T cells were incubated in a lysis solution to obtain raw cell extracts. Cellular soups of each extraction were then pooled, homogenized, and aliquoted to be further treated by three different protein-specific purification methods including acetone, acetone-methanol, and trichloroacetic acid (TCA)-acetone, each in triplicates. All the purified protein pellets were then dissolved in a standard rehydration buffer solution, loaded into the 17-cm immobilized pH gradient (IPG) strips, and separated according to their isoelectric points. Proteins were then separated once more according to their molecular weights in an O'Farrell separation system. Finally, by the visualization of the protein spots on the 2-dimentional profiles, quality and quantity of these 2-dimentional proteome patterns were then analyzed using the ImageMaster software.Findings: The obtained proteome recovery yields and total protein counts for acetone, acetone-methanol, and trichloroacetic acid-acetone methods were 0.100 ± 0.001, 0.070 ± 0.002, and 0.120 ± 0.005 ng/cell, and 1299 ± 9, 1698 ± 14 and 1973 ± 17, respectively. The results represent data obtained from three independent experiments.Conclusion: Trichloroacetic acid-acetone purification not only represented the highest recovery yield, suitable for expensive assays, but also showed the most suitable proteome coverage. So, the method is recommended for the comparative proteomic studies. However, the acetone-methanol procedure is more recommended for serological proteome analysis (SERPA); since it represents stronger protein spots which are more fitted to the immunoblotting procedure.

Keywords

References

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Journal of Isfahan Medical School
Volume 36, Issue 471 - Serial Number 471
January and February 2018
Pages 216-220

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History

  • Receive Date: 24 January 2018
  • Revise Date: 01 November 2024
  • Accept Date: 06 April 2022

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