Effect of polyethylene glycol 20 000 on protein extraction efficiency of formalin-fixed paraffin-embedded tissues in South Africa.
SP3-on-bead-digestion
archival tissue
formalin-fixed paraffin-embedded proteomics
mass spectrometry
polyethylene glycol 20 000
protein extraction
Journal
African journal of laboratory medicine
ISSN: 2225-2002
Titre abrégé: Afr J Lab Med
Pays: South Africa
ID NLM: 101603205
Informations de publication
Date de publication:
2021
2021
Historique:
received:
25
11
2019
accepted:
08
09
2021
entrez:
30
12
2021
pubmed:
31
12
2021
medline:
31
12
2021
Statut:
epublish
Résumé
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. 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. 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. 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. 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.
Sections du résumé
BACKGROUND
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
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
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
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
CONCLUSIONS
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.
Identifiants
pubmed: 34966662
doi: 10.4102/ajlm.v10i1.1122
pii: AJLM-10-1122
pmc: PMC8689371
doi:
Types de publication
Journal Article
Langues
eng
Pagination
1122Informations de copyright
© 2021. The Authors.
Déclaration de conflit d'intérêts
The authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article.
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