Original Research

Effect of polyethylene glycol 20 000 on protein extraction efficiency of formalin-fixed paraffin-embedded tissues in South Africa

Sophia Rossouw, Hocine Bendou, Liam Bell, Jonathan Rigby, Alan Christoffels
African Journal of Laboratory Medicine | Vol 10, No 1 | a1122 | DOI: https://doi.org/10.4102/ajlm.v10i1.1122 | © 2021 Alan Christoffels, Sophia Rossouw, Hocine Bendou, Liam Bell, Jonathan Rigby | This work is licensed under CC Attribution 4.0
Submitted: 25 November 2019 | Published: 17 December 2021

About the author(s)

Sophia Rossouw, South African Medical Research Council Bioinformatics Unit, South African National Bioinformatics Institute, University of the Western Cape, Cape Town, South Africa
Hocine Bendou, South African Medical Research Council Bioinformatics Unit, South African National Bioinformatics Institute, University of the Western Cape, Cape Town, South Africa
Liam Bell, Centre for Proteomic and Genomic Research, Observatory, Cape Town, South Africa
Jonathan Rigby, Department of Anatomical Pathology, National Health Laboratory Service, Tygerberg Hospital, Stellenbosch University, Cape Town, South Africa
Alan Christoffels, South African Medical Research Council Bioinformatics Unit, South African National Bioinformatics Institute, University of the Western Cape, Cape Town, South Africa

Abstract

Background: Optimal protocols for efficient and reproducible protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues are not yet standardised and new techniques are continually developed and improved. The effect of polyethylene glycol (PEG) 20 000 on protein extraction efficiency has not been evaluated using human FFPE colorectal cancer tissues and there is no consensus on the protein extraction solution required for efficient, reproducible extraction.

Objective: The impact of PEG 20 000 on protein extraction efficiency, reproducibility and protein selection bias was evaluated using FFPE colonic tissue via liquid chromatography tandem mass spectrometry analysis.

Methods: This study was conducted from August 2017 to July 2019 using human FFPE colorectal carcinoma tissues from the Anatomical Pathology department at Tygerberg Hospital in South Africa. Samples were analysed via label-free liquid chromatography tandem mass spectrometry to determine the impact of using PEG 20 000 in the protein extraction solution. Data were assessed regarding peptide and protein identifications, method efficiency, reproducibility, protein characteristics and organisation relating to gene ontology categories.

Results: Polyethylene glycol 20 000 exclusion increased peptides and proteins identifications and the method was more reproducible compared to the samples processed with PEG 20 000. However, no differences were observed with regard to protein selection bias. We found that higher protein concentrations (> 10 µg) compromised the function of PEG.

Conclusion: This study indicates that protocols generating high protein yields from human FFPE tissues would benefit from the exclusion of PEG 20 000 in the protein extraction solution.


Keywords

mass spectrometry; formalin-fixed paraffin-embedded proteomics; archival tissue; protein extraction; polyethylene glycol 20 000; SP3-on-bead-digestion

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