Validation of protein carbonyl measurement: a multi-centre study.
Validation of protein carbonyl measurement: a multi-centre study.
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DOI：
10.1016/j.redox.2014.12.014
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发表时间：
2015
期刊：
影响因子：
11.4
通讯作者：
Griffiths, Helen R.
中科院分区：
文献类型：
--
作者：
Augustyniak, Edyta;Adam, Aisha;Wojdyla, Katarzyna;Rogowska-Wrzesinska, Adelina;Willetts, Rachel;Korkmaz, Ayhan;Atalay, Mustafa;Weber, Daniela;Grune, Tilman;Borsa, Claudia;Gradinaru, Daniela;Bollineni, Ravi Chand;Fedorova, Maria;Griffiths, Helen R.
关键词：
蛋白质羰基被广泛分析作为蛋白质氧化的量度。有几种不同的方法来测定它们。先前的一项研究已经描述了当在单个实验室中使用不同的商业试剂盒通过ELISA分析蛋白质羰基时存在的数量级差异。我们进一步探讨了环研究中羰基分析差异的潜在原因。从大鼠肝脏制备可溶性蛋白级分，并暴露于0、5和15分钟的UV照射。冻干制剂被分发到六个不同的实验室，这些实验室在欧洲各地进行蛋白质羰基分析。ELISA和蛋白质印迹技术检测到增加蛋白质羰基形成0和5分钟之间的UV照射，无论使用的方法。照射15分钟后，少氧化检测到的实验室比5分钟照射后的一半。4个ELISA羰基结果中有3个在95%置信区间内。计算绝对羰基值时可能出现的错误可能归因于标准化的差异。在胰蛋白酶裂解辐照和对照肝蛋白后，多达88种蛋白质被鉴定为含有羰基，只有7种在所有三种肝制剂中是常见的。被羰基修饰的赖氨酸和精氨酸残基可能对胰蛋白酶的蛋白水解具有抗性。使用蛋白酶混合物可以增加氧化肽的回收。总之，标准化对于羰基分析至关重要，任何现有技术都无法有效分析重度氧化的蛋白质。蛋白质羰基的ELISA对于检测中度氧化具有灵敏度和耐用性。蛋白质羰基的蛋白质印迹法提供了一种稳健的半定量替代方法。蛋白质羰基分析方法需要改进标准化。质谱法适用于蛋白质氧化靶点的发现。相对于中度氧化的蛋白质，在重度氧化的蛋白质中检测到较少的羰基。
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. 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.
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影响因子：
10.6
作者：
Schaur, R. J.
通讯作者：
Schaur, R. J.
影响因子：
7.4
作者：
SHACTER, E;WILLIAMS, JA;LEVINE, RL
通讯作者：
LEVINE, RL
影响因子：
4.4
作者：
Bollineni, Ravi Chand;Fedorova, Maria;Hoffmann, Ralf
通讯作者：
Hoffmann, Ralf
影响因子：
3.5
作者：
GRIFFITHS, HR;LUNEC, J;WILLSON, RL
通讯作者：
WILLSON, RL
DOI：
10.1016/j.jchromb.2010.08.004
发表时间：
2011-05-15
影响因子：
3
作者：
Yan, Liang-Jun;Forster, Michael J.
通讯作者：
Forster, Michael J.