The aim of this study was to develop and validate the novel microLC/MS-MRM method for the simultaneous quantification of six
proteins:
angiopoietin 2 (Angpt-2), soluble form of
fms-like tyrosine kinase 1 (sFLT-1),
plasminogen activator inhibitor 1 (PAI-1),
tissue plasminogen activator (t-PA), endocan (ESM-1), soluble form of
E-selectin (sE-sel), and one
peptide:
adrenomedullin (ADM) in mouse plasma. Two approaches were compared: a stable
isotope dilution (
SID) method- used as a reference and a modified
SID (mSID) procedure. In
SID strategy the calibration curves were used, whereas in mSID the ratio between the chromatogram peak area of endogenous tryptic
peptides at unknown concentration to chromatogram peak area of exogenous, stable
isotope-labelled internal standards (SISs) added to the sample at known concentration was calculated. The microLC/MS-MRM method in the
SID approach was linear from 0.250 pmol/mL to 250 pmol/mL for Angpt-2; 5 pmol/mL to 5000 pmol/mL for sFLT-1; 2.5 pmol/mL to 5000 pmol/mL for PAI-1; 0.375 pmol/mL to 250 pmol/mL for t-PA; 0.375 pmol/mL to 187.5 pmol/mL for ESM-1; 2.5 pmol/mL to 5000 pmol/mL for sE-sel and 0.375 pmol/mL to 250 pmol/mL for ADM. LPS-induced changes in plasma assessed based on
SID and mSID approaches gave comparable quantitative results and featured LPS-induced dysregulation of endothelial permeability (Angpt-2, sFLT-1), glycocalyx injury (SDC-1) accompanied by a pro-thrombotic response (PAI-1). In addition, we applied microLC/MS-MRM method with mSID strategy to analyze human plasma samples from patients with
chronic myeloid leukemia (CML) and obstructive sleep apnoea (OSA) and demonstrated usefulness of the method to characterize endothelial function in humans. In conclusion, the microLC/MS-MRM method with mSID strategy applied for simultaneous quantification of
protein biomarkers of endothelial function in plasma represents a novel targeted proteomic platform for the comprehensive evaluation of endothelial function in mice and humans.