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.
Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics, Bobrzynskiego 14, 30-348 Krakow, Poland.
Talanta. 2019 Mar 1;194:1005-1016. doi: 10.1016/j.talanta.2018.10.067. Epub 2018 Oct 23.
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.
本研究旨在开发和验证一种新型的微 LC/MS-MRM 方法，用于同时定量检测六种蛋白质：血管生成素 2（Angpt-2）、可溶性 fms 样酪氨酸激酶 1（sFLT-1）、纤溶酶原激活物抑制剂 1（PAI-1）、组织型纤溶酶原激活物（t-PA）、内皮细胞特异性分子-1（Endocan，ESM-1）、可溶性 E-选择素（sE-sel）和一种肽：肾上腺髓质素（ADM）在小鼠血浆中。比较了两种方法：稳定同位素稀释（SID）法——用作参考和改良的 SID（mSID）程序。在 SID 策略中使用校准曲线，而在 mSID 中，计算未知浓度内源性胰蛋白酶肽的色谱峰面积与添加到样品中已知浓度的外源性稳定同位素标记内标（SISs）的色谱峰面积之间的比值。SID 方法中的微 LC/MS-MRM 方法对于 Angpt-2 为 0.250 pmol/mL 至 250 pmol/mL；sFLT-1 为 5 pmol/mL 至 5000 pmol/mL；PAI-1 为 2.5 pmol/mL 至 5000 pmol/mL；t-PA 为 0.375 pmol/mL 至 250 pmol/mL；ESM-1 为 0.375 pmol/mL 至 187.5 pmol/mL；sE-sel 为 2.5 pmol/mL 至 5000 pmol/mL，ADM 为 0.375 pmol/mL 至 250 pmol/mL。基于 SID 和 mSID 方法评估 LPS 诱导的血浆变化给出了可比的定量结果，并显示 LPS 诱导的内皮通透性紊乱（Angpt-2、sFLT-1）、糖萼损伤（SDC-1）伴随着促血栓反应（PAI-1）。此外，我们应用微 LC/MS-MRM 方法和 mSID 策略分析了慢性髓细胞白血病（CML）和阻塞性睡眠呼吸暂停（OSA）患者的人血浆样本，并证明了该方法在人类内皮功能特征中的有用性。总之，应用 mSID 策略的微 LC/MS-MRM 方法用于同时定量检测血浆内皮功能的蛋白质生物标志物，代表了一种新型的靶向蛋白质组学平台，用于全面评估小鼠和人类的内皮功能。
