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Sensitive, precise and accurate quantification of endothelial proteins in mouse plasma.A method used to evaluate protein profile in plasma of mice fed high–fat diet.A methodology that can be used to measure plasma protein concentrations in humans.The objective of this study was to develop and validate the method based on micro–liquid chromatography–tandem mass spectrometry (microLC/MS–MRM) for simultaneous determination of adiponectin (ADN), von Willebrand factor (vWF), soluble form of vascular cell adhesion molecule 1 (sVCAM–1), soluble form of intercellular adhesion molecule 1 (sICAM–1) and syndecan–1 (SDC–1) in mouse plasma. The calibration range was established from 2.5 pmol/mL to 5000 pmol/mL for ADN; 5 pmol/mL to 5000 pmol/mL for vWF; 0.375 pmol/mL to 250 pmol/mL for sVCAM–1 and sICAM–1; and 0.25 pmol/mL to 250 pmol/mL for SDC–1. The method was applied to measure the plasma concentration of selected proteins in mice fed high–fat diet (HFD), and revealed the pro–thrombotic status by increased concentration of vWF (1.31 ± 0.17 nmol/mL (Control) vs 1.98 ± 0.09 nmol/mL (HFD), p < 0.05) and the dysregulation of adipose tissue metabolism by decreased concentration of ADN (0.62 ± 0.08 nmol/mL (Control) vs 0.37 ± 0.06 nmol/mL (HFD), p < 0.05). In conclusion, the microLC/MS–MRM–based method allows for reliable measurements of selected protein biomarkers of endothelial dysfunction in mouse plasma.