• Sanja Radman, Sanda Raić, Ivona Bućan, Ajka Pribisalić, Josipa Dunatov, Ivana Mudnić, Mladen Boban, Francois Xavier Pellay, Ivana Kolčić, and Ozren Polašek.
• Ozren Polašek, University of Split School of Medicine, Šoltanska 2, 21000 Split, Croatia, ozren.polasek@mefst.hr.
• Croat. Med. J. 2020 Apr 30; 61 (2): 119-125.

AimTo develop a method for measuring protein carbonylation in human plasma and serum samples, which was previously implied in numerous age-related phenotypes.MethodsProtein expression and carbonylation were analyzed in plasma samples obtained from 12 healthy human individuals by using a novel method that combines affinity-based albumin and immunoglobulin G removal, and aminooxy dyeing in one- or two-dimensional gels. In addition, carbonylome profile of plasma and serum was compared. Coefficients of variation and intra-class correlation coefficients were used in statistical analysis.ResultsFollowing a step-wise laboratory development and optimization process, we measured the protein expression and carbonylation for 813 proteins from the plasma. The analysis of repeated measurements suggested excellent coefficients of variation, which rarely exceeded 10%. The average value of intra-class correlation based on absolute agreement (ICC) for protein expression was 0.97±0.02, while for carbonylation it was 0.73±0.24. The removal of the most extreme protein outlier in carbonylation assessment increased the average ICC to 0.87±0.04. Low protein spot volume substantially reduced repeatability. Serum carbonylation estimates were similar to those from plasma, with the ICC in the range of 0.86-0.89.ConclusionWe developed a reliable method for the measurement of human plasma protein carbonylation, which can be used for the assessment of carbonylome biomarkers of aging.

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