Original scientific paper
https://doi.org/10.3325/cmj.2020.61.119
Searching for carbonylome biomarkers of aging –development and validationof the proteomic method for quantification of carbonylated protein in human plasma
Sanja Radman
; Mediterranean Institute for Life Science, Split, Croatia
Sanda Raić
; University of Split School of Medicine, Split, Croatia
Ivona Bućan
; Mediterranean Institute for Life Science, Split, Croatia
Ajka Pribisalić
; Mediterranean Institute for Life Science, Split, Croatia
Josipa Dunatov
; University of Split School of Medicine, 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
orcid.org/0000-0001-7918-6052
; 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 carbo-
nylation in human plasma and serum samples, which was
previously implied in numerous age-related phenotypes.
Methods Protein expression and carbonylation were ana-
lyzed in plasma samples obtained from 12 healthy human
individuals by using a novel method that combines affin-
ity-based albumin and immunoglobulin G removal, and
aminooxy dyeing in one- or two-dimensional gels. In addi-
tion, carbonylome profile of plasma and serum was com-
pared. 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 ex-
pression and carbonylation for 813 proteins from the plas-
ma. 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 carbonyla-
tion assessment increased the average ICC to 0.87 ± 0.04.
Low protein spot volume substantially reduced repeatabil-
ity. 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 mea-
surement of human plasma protein carbonylation, which
can be used for the assessment of carbonylome biomark-
ers of aging.
Keywords
Hrčak ID:
253146
URI
Publication date:
14.6.2020.
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