Simultaneous Mass Spectrometry-Based Apolipoprotein Profiling and Apolipoprotein E Phenotyping in Patients with ASCVD and Mild Cognitive Impairment

Apolipoprotein E (apoE) occurs on the majority of plasma lipoproteins and plays a major role in the lipid metabolism in the periphery and in the central nervous system. ApoE is a polymorphic protein with three common isoforms, apoE2, apoE3 and apoE4, derived from respective alleles '2, '3 and '4. The aim of this study was to develop a sample pretreatment protocol combined with rapid mass spectrometry (MS)-based assay for simultaneous apolipoprotein profiling and apoE phenotype identification. This assay was validated in 481 samples from patients with stable atherosclerotic cardiovascular disease (ASCVD) and applied to study association with mild cognitive impairment (MCI) in the LIFE Adult study, including overall 690 study subjects. Simultaneous quantification of 8–12 major apolipoproteins including apoA-I, apoB-100 and apoE could be performed within 6.5 min. Phenotyping determined with the developed MS assay had good agreement with the genotyping by real-time fluorescence PCR (97.5%). ApoE2 isoform was associated with the highest total apoE concentration compared to apoE3 and apoE4 (p < 0.001). In the subgroup of diabetic atherosclerotic cardiovascular disease (ASCVD) patients, apoE2 isoform was related to higher apoC-I levels (apoE2 vs. apoE3, p < 0.05), while in the subgroup of ASCVD patients under statin therapy apoE2 was related to lower apoB-100 levels (apoE2 vs. apoE3/apoE4, p < 0.05). A significant difference in apoE concentration observed between mild cognitive impairment (MCI) subjects and controls was confirmed for each apoE phenotype. In conclusion, this study provides evidence for the successful implementation of an MS-based apoE phenotyping assay, which can be used to assess phenotype effects on plasma lipid and apolipoprotein levels

Development of a chiral SFC-MS/MS and reversed-phase LC-MS/MS platform for the quantitative metabolic profiling of octadecanoid oxylipins

Octadecanoids are broadly defined as oxylipins (i.e., lipid mediators) derived from 18-carbon fatty acids. In contrast to the well-studied eicosanoids, there is a lack of analytical methods for octadecanoids, hampering further investigations in the field. We developed an integrated workflow combining chiral separation by supercritical fluid chromatography (SFC) and reversed-phase liquid chromatography (LC) coupled to tandem-MS detection for quantification of a broad panel of octadecanoids. The platform included 70 custom-synthesized analytical and internal standards to extend the coverage of the octadecanoid synthetic pathways. A total of 103 octadecanoids could be separated by chiral SFC and complex enantioseparations could be performed in 0.995) and LLOQ ranged from 0.03-6.00 ng/mL for SFC and 0.01-1.25 ng/mL for LC. The average accuracy in solvent and surrogate matrix ranged from 89-109% in SFC and from 106-220% in LC, whereas coefficients of variation (CV) were <14% (at medium and high concentration) and 26% (at low concentration). Validation in surrogate matrix showed negligible matrix effects (<16% for all analytes) and average recoveries ranged from 71-83%. The combined methods provide a platform to investigate the biological activity of octadecanoids and expand our understanding of these little studied compounds

DATASET Comparative and Integrated Analysis of Plasma Extracellular Vesicles Isolation Methods in Healthy Volunteers and Patients Following Myocardial Infarction - Healthy Volunteer Dataset used for Integrated Analysis

The file contains data from the manuscript: Comparative and Integrated Analysis of Plasma Extracellular Vesicles Isolations Methods in Healthy Volunteers and Patients Following Myocardial Infarction. In short, plasma extracellular vesicles (EV) were isolated from 500 µL of platelet poor plasma of healthy volunteers and characterised by nanoparticle tracking analysis, protein concentration assay (BCA), targeted EV protein array, unbiased proteomics and targeted sphingolipidomics. The data was collated in an excel file and transformed to a Z-score. The data is deposited for public access

Comparative and integrated analysis of plasma extracellular vesicle isolation methods in healthy volunteers and patients following myocardial infarction

Plasma extracellular vesicle (EV) number and composition are altered following myocardial infarction (MI), but to properly understand the significance of these changes it is essential to appreciate how the different isolation methods affect EV characteristics, proteome and sphingolipidome. Here, we compared plasma EV isolated from platelet-poor plasma from four healthy donors and six MI patients at presentation and 1-month post-MI using ultracentrifugation (UC), polyethylene glycol precipitation, acoustic trapping, size-exclusion chromatography (SEC) and immunoaffinity capture. The isolated EV were evaluated by Nanoparticle Tracking Analysis (NTA), Western blot, transmission electron microscopy (TEM), an EV-protein array, untargeted proteomics (LC-MS/MS) and targeted sphingolipidomics (LC-MS/MS). The application of the five different plasma EV isolation methods in patients presenting with MI showed that the choice of plasma EV isolation method influenced the ability to distinguish elevations in plasma EV concentration following MI, enrichment of EV-cargo (EV-proteins and sphingolipidomics) and associations with the size of the infarct determined by cardiac magnetic resonance imaging 6 months post-MI. Despite the selection bias imposed by each method, a core of EV-associated proteins and lipids was detectable using all approaches. However, this study highlights how each isolation method comes with its own idiosyncrasies and makes the comparison of data acquired by different techniques in clinical studies problematic