title: Validation of protein carbonyl measurement: a multi-centre study.
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.
authors.affiliation: Life & Health Sciences, Aston University, Birmingham B4 7ET, UK.
authors.forename: Edyta
authors.initials: E
authors.lastname: Augustyniak
authors.affiliation: Life & Health Sciences, Aston University, Birmingham B4 7ET, UK.
authors.forename: Aisha
authors.initials: A
authors.lastname: Adam
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Denmark.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Wojdyla
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Denmark.
authors.forename: Adelina
authors.initials: A
authors.lastname: Rogowska-Wrzesinska
authors.affiliation: Life & Health Sciences, Aston University, Birmingham B4 7ET, UK.
authors.forename: Rachel
authors.initials: R
authors.lastname: Willetts
authors.affiliation: University of Eastern Finland, Institute of Biomedicine, Physiology, Finland.
authors.forename: Ayhan
authors.initials: A
authors.lastname: Korkmaz
authors.affiliation: Institute of Biomedicine, Physiology, University of Kuopio, Kuopio, Finland.
authors.forename: Mustafa
authors.initials: M
authors.lastname: Atalay
authors.affiliation: German Institute of Human Nutrition, DIfE, Nuthetal 14558, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Weber
authors.affiliation: German Institute of Human Nutrition, DIfE, Nuthetal 14558, Germany.
authors.forename: Tilman
authors.initials: T
authors.lastname: Grune
authors.affiliation: Ana Aslan - National Institute of Gerontology and Geriatrics, PO Box 2-4, Bucharest 011241, Romania.
authors.forename: Claudia
authors.initials: C
authors.lastname: Borsa
authors.affiliation: Faculty of Pharmacy, Department of Biochemistry, Carol Davila - University of Medicine and Pharmacy, Bucharest 020956, Romania.
authors.forename: Daniela
authors.initials: D
authors.lastname: Gradinaru
authors.affiliation: Faculty of Chemistry and Mineralogy, Center for Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, Universität Leipzig, Leipzig, Germany.
authors.forename: Ravi
authors.initials: R
authors.lastname: Chand Bollineni
authors.affiliation: Faculty of Chemistry and Mineralogy, Center for Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, Universität Leipzig, Leipzig, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Fedorova
authors.affiliation: Life & Health Sciences, Aston University, Birmingham B4 7ET, UK. Electronic address: h.r.griffiths@aston.ac.uk.
authors.forename: Helen R
authors.initials: HR
authors.lastname: Griffiths
mesh_terms: D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D013058:Mass Spectrometry; D010084:Oxidation-Reduction / Q000528:radiation effects*; D050050:Protein Carbonylation / Q000528:radiation effects*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D014466:Ultraviolet Rays
chemical_list: D011506:Proteins; C074188:O-(biotinylcarbazoylmethyl)hydroxylamine; D001710:Biotin
keywords: Aldehyde reactive probe; Carbonyl ELISA; Mass spectrometry; Oxidised protein Western blot; Protein oxidation