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Cited by 135Redox biology and oxidative stressAntioxidant Activity and Oxidative StressAdvanced Glycation End Products research

Validation of protein carbonyl measurement: A multi-centre study

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Augustyniak, Edyta, Adam, Aisha, Wojdyla, Katarzyna, Rogowska-Wrzesinska, Adelina, Willetts, Rachel, Korkmaz, Ayhan, Atalay, Mustafa, Weber, Daniela +6 more·2015·Redox Biology
DOI 10.1016/j.redox.2014.12.014PMC PMC4309846
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Abstract

Protein carbonyls are widely analysed as a measure of protein oxidation. Several different methods exist for their determination. A previous study had described orders of magnitude variance that existed when protein carbonyls were analysed in a single laboratory by ELISA using different commercial kits. We have further explored the potential causes of variance in carbonyl analysis in a ring study. A soluble protein fraction was prepared from rat liver and exposed to 0, 5 and 15min of UV irradiation. Lyophilised preparations were distributed to six different laboratories that routinely undertook protein carbonyl analysis across Europe. ELISA and Western blotting techniques detected an increase in protein carbonyl formation between 0 and 5min of UV irradiation irrespective of method used. After irradiation for 15min, less oxidation was detected by half of the laboratories than after 5min irradiation. Three of the four ELISA carbonyl results fell within 95% confidence intervals. Likely errors in calculating absolute carbonyl values may be attributed to differences in standardisation. Out of up to 88 proteins identified as containing carbonyl groups after tryptic cleavage of irradiated and control liver proteins, only seven were common in all three liver preparations. Lysine and arginine residues modified by carbonyls are likely to be resistant to tryptic proteolysis. Use of a cocktail of proteases may increase the recovery of oxidised peptides. In conclusion, standardisation is critical for carbonyl analysis and heavily oxidised proteins may not be effectively analysed by any existing technique.

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  • LC-MS/MS has been used for proteomic analysis to identify carbonylated proteins, providing a more sensitive approach, but reveals limitations for quantitative assessments.

    “Besides this, LC-MS/MS has been also used for proteomic analysis to identify carbonylated proteins, providing a more sensitive approach, but reveal limitations for quantitative assessments [86].”

    From Protein oxidation - Formation mechanisms, detection and relevance as biomarkers in human diseases (2021) · cites this paper as [86]

    The Results and discussion section reports using LC–MS/MS to identify carbonylated proteins and says the method used provided no quantitative information. The Conclusions likewise describe poorly developed quantitative applications for MS. The paper does not establish the claim that LC–MS/MS is a more sensitive approach. (LLM, conf=0.86)

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Authors
Augustyniak, E., Adam, A., Wojdyla, K., Rogowska-Wrzesinska, A., Willetts, R., Korkmaz, A., Atalay, M., Weber, D., Grune, T., Borsa, C., Gradinaru, D., Chand Bollineni, R., Fedorova, M., & Griffiths, H. R.
Affiliations
  • Aston University (GB)
  • University of Eastern Finland (FI)
  • German Institute of Human Nutrition (DE)
  • Leipzig University (DE)
Corresponding author
Griffiths, Helen R.
ORCID
Venue
Redox Biology
Publisher
Elsevier BV
Published
2015-04
Volume
4
Pages
149-157
ISSN
2213-2317 (ISSN-L)
DOI
10.1016/j.redox.2014.12.014
PMC
PMC4309846
OpenAlex
W1992902689
Cited by
135
References
2
Citations by year
2026: 5 · 2025: 11 · 2024: 12 · 2023: 6 · 2022: 8 · 2021: 19 · 2020: 19 · 2019: 17 · 2018: 12 · 2017: 7 · 2016: 13 · 2015: 6
FWCI
3.56
Type
journal article
Language
en
Open access
Licence
Locations
  • Redox Biology (publishedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Aston Publications Explorer (Aston University) (acceptedVersion)
  • UEF eRepo (University of Eastern Finland) (submittedVersion, cc-by-nc-nd)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Informationszentrum Lebenswissenschaften (ZB MED) (submittedVersion, cc-by-nc-nd)
  • University of Southern Denmark Research Portal (University of Southern Denmark) (submittedVersion, cc-by-nc-nd)
Funders
  • COST (CM1001)
  • European Fund for Regional Structure Development (100146238, 100121468)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
  • Health Sciences › Medicine › Biochemistry › Antioxidant Activity and Oxidative Stress
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Advanced Glycation End Products research
Keywords
protein carbonyls, protein oxidation, method validation, inter-laboratory variability, ELISA kit, UV irradiation, rat liver, standardization, tryptic proteolysis, multicenter study
MeSH terms
Animals, Biotin, Liver, Oxidation-Reduction, Proteins, Mass Spectrometry, Ultraviolet Rays, Protein Carbonylation, Rats

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