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Shotgun Proteomics on Tissue Specimens Extracted with Acid Guanidinium-Thiocyanate-Phenol-Chloroform

  • René B. H. Braakman
  • Anieta M. Sieuwerts
  • Arzu UmarEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1293)

Abstract

Protein-containing organic fractions of acid guanidinium thiocyanate-phenol-chloroform-extracted tissues are an interesting source of proteins as this method is widely used for RNA extraction for gene expression analysis. However, due to difficulties in redissolving pelleted proteins from the organic phase, protein analysis has only been limitedly reported. Current shotgun mass spectrometry-based methods, however, require minute amounts of sample, and methods have been developed that allow SDS to be removed from an extraction buffer prior to protein digestion. The limited volume of starting material needed for shotgun proteomics facilitates redissolving proteins in SDS-containing buffers, allowing proteins to be readily extracted. Here we describe a protocol for an SDS-DTT-based extraction of proteins from the organic fraction of acid guanidinium-thiocyanate-phenol-chloroform-extracted tissues that remain after RNA isolation for shotgun MS analysis.

Key words

Breast cancer Sample preparation Proteomics Genomics Acid guanidinium-thiocyanate-phenol-chloroform extraction 

Notes

Acknowledgements

This work was financially supported through the Center for Translational Molecular Medicine, Breast CARE (03O-104-06).

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Copyright information

© Springer Science+Business Media LLC New York 2015

Authors and Affiliations

  • René B. H. Braakman
    • 1
    • 2
    • 3
  • Anieta M. Sieuwerts
    • 1
    • 2
    • 3
  • Arzu Umar
    • 2
    • 3
    • 4
    Email author
  1. 1.Department of Medical Oncology, Erasmus MC Cancer InstituteUniversity Medical Center RotterdamRotterdamThe Netherlands
  2. 2.Postgraduate School of Molecular MedicineErasmus University Medical CenterRotterdamThe Netherlands
  3. 3.Center for Translational Molecular MedicineEindhovenThe Netherlands
  4. 4.Laboratory of Breast Cancer Genomics and Proteomics, Department of Medical Oncology, Erasmus MC Cancer InstituteUniversity Medical Center RotterdamRotterdamThe Netherlands

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