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Searching for carbonylome biomarkers of aging - development and validation of the proteomic method for quantification of carbonylated protein in human plasma.

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.

SUBMITTER: Radman S

PROVIDER: S-EPMC7230409 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Searching for carbonylome biomarkers of aging - development and validation of the proteomic method for quantification of carbonylated protein in human plasma.

Radman Sanja S Raić Sanda S Bućan Ivona I Pribisalić Ajka A Dunatov Josipa J Mudnić Ivana I Boban Mladen M Pellay Francois Xavier FX Kolčić Ivana I Polašek Ozren O

Croatian medical journal 20200401 2

<h4>Aim</h4>To develop a method for measuring protein carbonylation in human plasma and serum samples, which was previously implied in numerous age-related phenotypes.<h4>Methods</h4>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 comp ...[more]

PMID: 32378378

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Project description:A fast, accurate and reliable ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for simultaneous quantification of ivermectin (IVER), doramectin (DORA), and moxidectin (MOXI) in bovine plasma. A priority for sample preparation was the eradication of possible infectious diseases to avoid travel restrictions. The sample preparation was based on protein precipitation using 1% formic acid in acetonitrile, followed by Ostro® 96-well plate pass-through sample clean-up. The simple and straightforward procedure, along with the short analysis time, makes the current method unique and suitable for a large set of sample analyses per day for PK studies. Chromatographic separation was performed using an Acquity UPLC HSS-T3 column, with 0.01% acetic acid in water and methanol, on an Acquity H-Class ultra-high performance liquid chromatograph (UHPLC) system. The MS/MS instrument was a Xevo TQ-S® mass spectrometer, operating in the positive electrospray ionization mode and two multiple reaction monitoring (MRM) transitions were monitored per component. The MRM transitions of m/z 897.50 > 753.4 for IVER, m/z 921.70 > 777.40 for DORA and m/z 640.40 > 123.10 for MOXI were used for quantification. The method validation was performed using matrix-matched calibration curves in a concentration range of 1 to 500 ng/mL. Calibration curves fitted a quadratic regression model with 1/x2 weighting (r ≥ 0.998 and GoF ≤ 4.85%). Limits of quantification (LOQ) values of 1 ng/mL were obtained for all the analytes, while the limits of detection (LOD) were 0.02 ng/mL for IVER, 0.03 ng/mL for DORA, and 0.58 ng/mL for MOXI. The results of within-day (RSD < 6.50%) and between-day (RSD < 8.10%) precision and accuracies fell within acceptance ranges. No carry-over and no peak were detected in the UHPLC-MS/MS chromatogram of blank samples showing good specificity of the method. The applicability of the developed method was proved by an analysis of the field PK samples.

Dataset Information

Searching for carbonylome biomarkers of aging - development and validation of the proteomic method for quantification of carbonylated protein in human plasma.

Publications

Searching for carbonylome biomarkers of aging - development and validation of the proteomic method for quantification of carbonylated protein in human plasma.

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