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 and Griffiths, Helen (2015). Validation of protein carbonyl measurement:a multi-centre study. Redox biology, 4 , 149–157.

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.

Publication DOI: https://doi.org/10.1016/j.redox.2014.12.014
Divisions: Life & Health Sciences
Life & Health Sciences > Biosciences
Additional Information: 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)
Uncontrolled Keywords: Aldehyde reactive probe,Carbonyl ELISA,Mass spectrometry,Oxidised protein western blot,Protein oxidation,Biochemistry,Organic Chemistry
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Related URLs: http://www.scopus.com/inward/record.url?scp=84920386346&partnerID=8YFLogxK (Scopus URL)
Published Date: 2015-05
Authors: 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 ( 0000-0002-2666-2147)

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