Validation of protein carbonyl measurement: a multi-centre study

Edyta Augustyniak, Aisha Adam, Katarzyna Wojdyla, Adelina Rogowska-Wrzesinska, Rachel Willetts, Ayhan Korkmaz, Mustafa Atalay, Daniela Weber, Tilman Grune, Claudia Borsa, Daniela Gradinaru, Ravi Chand Bollineni, Maria Fedorova, Helen Griffiths

Research output: Contribution to journalArticle

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 15 min 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 5 min of UV irradiation irrespective of method used. After irradiation for 15 min, less oxidation was detected by half of the laboratories than after 5 min 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.
LanguageEnglish
Pages149–157
Number of pages9
JournalRedox biology
Volume4
Early online date24 Dec 2014
DOIs
Publication statusPublished - May 2015

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Proteins
Irradiation
Liver
Enzyme-Linked Immunosorbent Assay
Standardization
Protein Carbonylation
Proteolysis
Oxidation
Lysine
Arginine
Peptide Hydrolases
Rats
Western Blotting
Confidence Intervals
Peptides
Recovery

Bibliographical note

Creative Commons: Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)

Funding: COSTCM1001. Financial support from the European Fund for Regional Structure Development (EFRE, European Union and Free State Saxony; 100146238 and 100121468 to M.F)

Keywords

  • Aldehyde reactive probe
  • Carbonyl ELISA
  • Mass spectrometry
  • Oxidised protein western blot
  • Protein oxidation

Cite this

Augustyniak, E., Adam, A., Wojdyla, K., Rogowska-Wrzesinska, A., Willetts, R., Korkmaz, A., ... Griffiths, H. (2015). Validation of protein carbonyl measurement: a multi-centre study. Redox biology, 4, 149–157. https://doi.org/10.1016/j.redox.2014.12.014
Augustyniak, Edyta ; Adam, Aisha ; Wojdyla, Katarzyna ; Rogowska-Wrzesinska, Adelina ; Willetts, Rachel ; Korkmaz, Ayhan ; Atalay, Mustafa ; Weber, Daniela ; Grune, Tilman ; Borsa, Claudia ; Gradinaru, Daniela ; Chand Bollineni, Ravi ; Fedorova, Maria ; Griffiths, Helen. / Validation of protein carbonyl measurement : a multi-centre study. In: Redox biology. 2015 ; Vol. 4. pp. 149–157.
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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 2015, 'Validation of protein carbonyl measurement: a multi-centre study' Redox biology, vol. 4, pp. 149–157. https://doi.org/10.1016/j.redox.2014.12.014

Validation of protein carbonyl measurement : a multi-centre study. / Augustyniak, Edyta; Adam, Aisha; Wojdyla, Katarzyna; Rogowska-Wrzesinska, Adelina; Willetts, Rachel; Korkmaz, Ayhan; Atalay, Mustafa; Weber, Daniela; Grune, Tilman; Borsa, Claudia; Gradinaru, Daniela; Chand Bollineni, Ravi; Fedorova, Maria; Griffiths, Helen.

In: Redox biology, Vol. 4, 05.2015, p. 149–157.

Research output: Contribution to journalArticle

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AU - Rogowska-Wrzesinska, Adelina

AU - Willetts, Rachel

AU - Korkmaz, Ayhan

AU - Atalay, Mustafa

AU - Weber, Daniela

AU - Grune, Tilman

AU - Borsa, Claudia

AU - Gradinaru, Daniela

AU - Chand Bollineni, Ravi

AU - Fedorova, Maria

AU - Griffiths, Helen

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Augustyniak E, Adam A, Wojdyla K, Rogowska-Wrzesinska A, Willetts R, Korkmaz A et al. Validation of protein carbonyl measurement: a multi-centre study. Redox biology. 2015 May;4:149–157. https://doi.org/10.1016/j.redox.2014.12.014