Validation of protein carbonyl measurement: A multi-centre study
| Autor: | Tilman Grune, Mustafa Atalay, Daniela Weber, Ravi Chand Bollineni, Daniela Gradinaru, Claudia Borsa, Rachel S. Willetts, Ayhan Korkmaz, Aisha Adam, Adelina Rogowska-Wrzesinska, Edyta Augustyniak, Helen R. Griffiths, Katarzyna Wojdyla, Maria Fedorova |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
Proteases
Arginine Ultraviolet Rays Protein Carbonylation Proteolysis Clinical Biochemistry Lysine Biotin Aldehyde reactive probe Protein oxidation Biochemistry chemistry.chemical_compound Oxidised protein Western blot medicine Animals Carbonyl ELISA lcsh:QH301-705.5 lcsh:R5-920 Chromatography Mass spectrometry medicine.diagnostic_test Organic Chemistry Proteins Orders of magnitude (mass) Rats lcsh:Biology (General) Liver chemistry Western blot Oxidised protein lcsh:Medicine (General) Oxidation-Reduction Research Paper |
| Zdroj: | Redox Biology 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 2015, ' Validation of protein carbonyl measurement : A multi-centre study ', Redox Biology, vol. 4C, pp. 149-157 . https://doi.org/10.1016/j.redox.2014.12.014 Redox Biology, 4: 149-157 Redox Biology, Vol 4, Iss C, Pp 149-157 (2015) |
| ISSN: | 2213-2317 |
| DOI: | 10.1016/j.redox.2014.12.014 |
| Popis: | 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. Graphical abstract Highlights • ELISA of protein carbonyls is sensitive and robust to detect moderate oxidation. • Western blot for protein carbonyls offers a robust semi-quantitative alternative. • Improved standardisation is needed for protein carbonyl analysis methods. • Mass spectrometry is suitable for protein oxidation target discovery. • Fewer carbonyls are detected in heavily-relative to moderately-oxidised proteins. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|