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Talanta. 2019 Mar 1;194:1005-1016. doi: 10.1016/j.talanta.2018.10.067. Epub 2018 Oct 23.

Quantitative measurement of selected protein biomarkers of endothelial dysfunction in plasma by micro-liquid chromatography-tandem mass spectrometry based on stable isotope dilution method.

Author information

1
Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics, Bobrzynskiego 14, 30-348 Krakow, Poland; Jagiellonian University Medical College, Faculty of Pharmacy, Chair and Department of Toxicology, Medyczna 9, 30-688 Krakow, Poland.
2
Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics, Bobrzynskiego 14, 30-348 Krakow, Poland.
3
Jagiellonian University Medical College, Faculty of Pharmacy, Chair and Department of Toxicology, Medyczna 9, 30-688 Krakow, Poland.
4
Jagiellonian University Medical College, Faculty of Medicine, Chair and Department of Haematology, Kopernika 17, 31-501 Krakow, Poland.
5
Jagiellonian University Medical College, Faculty of Medicine, Department of Pulmonology, II Chair of Internal Medicine, Skawinska 8, 31-066 Krakow, Poland.
6
Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics, Bobrzynskiego 14, 30-348 Krakow, Poland; Jagiellonian University Medical College, Faculty of Medicine, Chair of Pharmacology, Grzegorzecka 16, 31-531 Krakow, Poland. Electronic address: stefan.chlopicki@jcet.eu.
7
Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics, Bobrzynskiego 14, 30-348 Krakow, Poland; Jagiellonian University Medical College, Faculty of Pharmacy, Chair and Department of Toxicology, Medyczna 9, 30-688 Krakow, Poland. Electronic address: maria.walczak@jcet.eu.

Abstract

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.

KEYWORDS:

Biomarkers; Endothelium; Mice; MicroLC/MS-MRM; SID; Targeted proteomics

PMID:
30609507
DOI:
10.1016/j.talanta.2018.10.067
[Indexed for MEDLINE]

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