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Improved in-solution trypsin digestion method for methanol-chloroform precipitated cellular proteomics sample.

A D A Shahinuzzaman1, Jayanta K Chakrabarty1, Zixiang Fang1

  • 1Department of Chemistry and Biochemistry, University of Texas, Arlington, Texas, USA.

Journal of Separation Science
|February 20, 2020
PubMed
Summary

This study introduces a new method for protein analysis using methanol-chloroform precipitation and deoxycholic acid. This improved sample preparation enhances protein identification in mass spectrometry-based proteomics.

Keywords:
deoxycholic acidmethanol-chloroform precipitationproteomics sample preparationraw macrophagestrypsin digestion

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Area of Science:

  • Proteomics
  • Mass Spectrometry
  • Biochemistry

Background:

  • Methanol-chloroform precipitation is crucial for cellular proteomics but protein re-solubilization is challenging.
  • Sodium deoxycholate aids in re-solubilizing membrane proteins.

Purpose of the Study:

  • To evaluate the efficacy of deoxycholic acid-assisted re-solubilization following methanol-chloroform precipitation for improved protein identification.
  • To compare this modified method against established sample digestion protocols in bottom-up proteomics.

Main Methods:

  • Methanol-chloroform precipitation of Raw 264.7 mouse macrophage cell lysate.
  • Deoxycholic acid treatment for re-solubilization of precipitated proteins.
  • In-solution trypsin digestion and liquid chromatography-tandem mass spectrometry analysis.

Main Results:

  • Deoxycholic acid treatment resulted in an 82% increase in protein identification compared to other methods.
  • Liquid chromatography-tandem mass spectrometry showed a 14% increase in protein identification and a 27% increase in unique protein identifications.
  • The modified method significantly improved protein recovery and identification from cell lysates.

Conclusions:

  • Deoxycholic acid-assisted re-solubilization is an effective strategy to overcome protein insolubility after methanol-chloroform precipitation.
  • This improved digestion method offers a valuable alternative for mammalian cell sample preparation in proteomics.
  • The findings are particularly relevant for samples prepared using sodium dodecyl sulfate-based lysis buffers.