Original scientific paper
https://doi.org/10.3325/cmj.2020.61.119
Searching for carbonylome biomarkers of aging – development and validation of the proteomic method for quantification of carbonylated protein in human plasma
Sanja Radman
; Mediterranean Institute for Life Science, Split, Croatia
Sanda Raić
; Mediterranean Institute for Life Science, Split, Croatia
Ivona Bućan
; University of Split School of Medicine, Split, Croatia
Ajka Pribisalić
; University of Split School of Medicine, Split, Croatia
Josipa Dunatov
; Mediterranean Institute for Life Science, Split, Croatia
Ivana Mudnić
; University of Split School of Medicine, Split, Croatia
Mladen Boban
; University of Split School of Medicine, Split, Croatia
Francois Xavier Pellay
; Mediterranean Institute for Life Science, Split, Croatia
Ivana Kolčić
; University of Split School of Medicine, Split, Croatia
Ozren Polašek
orcid.org/0000-0002-5765-1862
; University of Split School of Medicine, Split, Croatia
Abstract
Aim To develop a method for measuring protein carbonylation in human plasma and serum samples, which was previously implied in numerous age-related phenotypes.
Methods Protein 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. Results Following 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.
Conclusion We developed a reliable method for the measurement of human plasma protein carbonylation, which can be used for the assessment of carbonylome biomarkers of aging.
Keywords
Hrčak ID:
240163
URI
Publication date:
15.4.2020.
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