Journal
TALANTA
Volume 182, Issue -, Pages 492-499Publisher
ELSEVIER
DOI: 10.1016/j.talanta.2018.01.044
Keywords
Calcific aortic valve stenosis; Heme oxygenase-1; Ectonucleoside triphosphate diphosphohydrolase; Ecto-5 '-nucleotidase; Human aortic valves; Targeted proteomics
Categories
Funding
- TEAM program of Foundation for Polish Science [TEAM/2011-8/7]
- National Science Centre of Poland [2011/01/B/NZ4/03719, 2016/22/M/NZ4/00678]
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Several proteins such as membrane-associated ectonucleotidases: ecto-5'-nucleotidase (E5NT/CD73) and ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39), and intracellular heme oxygenase-1 (HO-1) may contribute to protection from inflammation-related diseases such as calcific aortic valve stenosis (CAS). Accurate quantification of these proteins could contribute to better understanding of the disease mechanisms and identification of biomarkers. This report presents development and validation of quantification method for E5NT/CD73, ENTPD1/CD39 and HO-1. The multiplexed targeted proteomic assay involved antibody-free, multiple-enzyme digestion, filter-assisted sample preparation (MED-FASP) strategy and a nanoflow liquid chromatography/mass spectrometry under multiple reaction monitoring mode (LC-MRM/MS). The method developed presented high sensitivity (LLOQ of 5 pg/mL for each of the analytes) and accuracy that ranged from 92.0% to 107.0%, and was successfully applied for the absolute quantification of HO-1, CD39 and CD73 proteins in homogenates of human calcified and non-calcified valves. The absolute CD39 and CD73 concentrations were lower in calcified aortic valves (as compared to non-stenotic ones) and were found to be: 1.16 +/- 0.39 vs. 3.15 +/- 0.37 pmol/mg protein and 1.94 +/- 0.21 vs. 2.39 +/- 0.39 pmol/mg protein, respectively, while the quantity of HO-1 was elevated in calcified valves (10.72 +/- 1.18 vs. 4.28 +/- 0.42 amol/mg protein). These results were consistent but more reproducible as compared to immunoassays. In conclusion, multiplexed quantification of HO-1, CD39 and CD73 proteins by LC-MRM/MS works well in challenging human tissues such as aortic valves. This analysis confirmed the relevance of these proteins in pathogenesis of CAS and could be extended to other biomedical investigations.
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