Effect of polyethylene glycol 20 000 on protein extraction efficiency of formalin-fixed paraffin-embedded tissues in South Africa
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...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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AOSIS
2021-12-01
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| Series: | African Journal of Laboratory Medicine |
| Subjects: | |
| Online Access: | https://ajlmonline.org/index.php/ajlm/article/view/1122 |
| _version_ | 1811301306726350848 |
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| author | Sophia Rossouw Hocine Bendou Liam Bell Jonathan Rigby Alan Christoffels |
| author_facet | Sophia Rossouw Hocine Bendou Liam Bell Jonathan Rigby Alan Christoffels |
| author_sort | Sophia Rossouw |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T07:06:20Z |
| format | Article |
| id | doaj.art-32b87c64340d46f0b5cb9c838289b5cb |
| institution | Directory Open Access Journal |
| issn | 2225-2002 2225-2010 |
| language | English |
| last_indexed | 2024-04-13T07:06:20Z |
| publishDate | 2021-12-01 |
| publisher | AOSIS |
| record_format | Article |
| series | African Journal of Laboratory Medicine |
| spelling | doaj.art-32b87c64340d46f0b5cb9c838289b5cb2022-12-22T02:56:59ZengAOSISAfrican Journal of Laboratory Medicine2225-20022225-20102021-12-01101e1e1010.4102/ajlm.v10i1.1122348Effect of polyethylene glycol 20 000 on protein extraction efficiency of formalin-fixed paraffin-embedded tissues in South AfricaSophia Rossouw0Hocine Bendou1Liam Bell2Jonathan Rigby3Alan Christoffels4South African Medical Research Council Bioinformatics Unit, South African National Bioinformatics Institute, University of the Western Cape, Cape TownSouth African Medical Research Council Bioinformatics Unit, South African National Bioinformatics Institute, University of the Western Cape, Cape TownCentre for Proteomic and Genomic Research, Observatory, Cape TownDepartment of Anatomical Pathology, National Health Laboratory Service, Tygerberg Hospital, Stellenbosch University, Cape TownSouth African Medical Research Council Bioinformatics Unit, South African National Bioinformatics Institute, University of the Western Cape, Cape TownBackground: 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.https://ajlmonline.org/index.php/ajlm/article/view/1122mass spectrometryformalin-fixed paraffin-embedded proteomicsarchival tissueprotein extractionpolyethylene glycol 20 000sp3-on-bead-digestion |
| spellingShingle | Sophia Rossouw Hocine Bendou Liam Bell Jonathan Rigby Alan Christoffels Effect of polyethylene glycol 20 000 on protein extraction efficiency of formalin-fixed paraffin-embedded tissues in South Africa African Journal of Laboratory Medicine mass spectrometry formalin-fixed paraffin-embedded proteomics archival tissue protein extraction polyethylene glycol 20 000 sp3-on-bead-digestion |
| title | Effect of polyethylene glycol 20 000 on protein extraction efficiency of formalin-fixed paraffin-embedded tissues in South Africa |
| title_full | Effect of polyethylene glycol 20 000 on protein extraction efficiency of formalin-fixed paraffin-embedded tissues in South Africa |
| title_fullStr | Effect of polyethylene glycol 20 000 on protein extraction efficiency of formalin-fixed paraffin-embedded tissues in South Africa |
| title_full_unstemmed | Effect of polyethylene glycol 20 000 on protein extraction efficiency of formalin-fixed paraffin-embedded tissues in South Africa |
| title_short | Effect of polyethylene glycol 20 000 on protein extraction efficiency of formalin-fixed paraffin-embedded tissues in South Africa |
| title_sort | effect of polyethylene glycol 20 000 on protein extraction efficiency of formalin fixed paraffin embedded tissues in south africa |
| topic | mass spectrometry formalin-fixed paraffin-embedded proteomics archival tissue protein extraction polyethylene glycol 20 000 sp3-on-bead-digestion |
| url | https://ajlmonline.org/index.php/ajlm/article/view/1122 |
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