This study applied mass spectrometry-based lipidomics profiling to population-based cohorts and identified molecular lipid signatures for cardiovascular disease. Molecular lipid species constitute promising new biomarkers that outperform the conventional biochemical measurements of lipid classes currently used in clinics.
Background-We sought to perform a systematic lipid analysis of atherosclerotic plaques using emerging mass spectrometry techniques. Methods and Results-A chip-based robotic nanoelectrospray platform interfaced to a triple quadrupole mass spectrometer was adapted to analyze lipids in tissue sections and extracts from human endarterectomy specimens by shotgun lipidomics. Eighteen scans for different lipid classes plus additional scans for fatty acids resulted in the detection of 150 lipid species from 9 different classes of which 24 were detected in endarterectomies only. Further analyses focused on plaques from symptomatic and asymptomatic patients and stable versus unstable regions within the same lesion. Polyunsaturated cholesteryl esters with long-chain fatty acids and certain sphingomyelin species showed the greatest relative enrichment in plaques compared to plasma and formed part of a lipid signature for vulnerable and stable plaque areas in a systems-wide network analysis. In principal component analyses, the combination of lipid species across different classes provided a better separation of stable and unstable areas than individual lipid classes.
Conclusions-This
BackgroundGlyoxalase 1 (Glo1) and glyoxalase 2 (Glo2) are ubiquitously expressed cytosolic enzymes that catalyze the conversion of toxic α-oxo-aldehydes into the corresponding α-hydroxy acids using L-glutathione (GSH) as a cofactor. Human Glo1 exists in various isoforms; however, the nature of its modifications and their distinct functional assignment is mostly unknown.Methodology/Principal FindingsWe characterized native Glo1 purified from human erythrocytes by mass spectrometry. The enzyme was found to undergo four so far unidentified posttranslational modifications: (i) removal of the N-terminal methionine 1, (ii) N-terminal acetylation at alanine 2, (iii) a vicinal disulfide bridge between cysteine residues 19 and 20, and (iv) a mixed disulfide with glutathione on cysteine 139. Glutathionylation of Glo1 was confirmed by immunological methods. Both, N-acetylation and the oxidation state of Cys19/20, did not impact enzyme activity. In contrast, glutathionylation strongly inhibited Glo1 activity in vitro. The discussed mechanism for enzyme inhibition by glutathionylation was validated by molecular dynamics simulation.Conclusion/SignificanceIt is shown for the first time that Glo1 activity directly can be regulated by an oxidative posttranslational modification that was found in the native enzyme, i.e., glutathionylation. Inhibition of Glo1 by chemical reaction with its co-factor and the role of its intramolecular disulfides are expected to be important factors within the context of redox-dependent regulation of glucose metabolism in cells.
The application of proteomics to identify MMP targets is a new frontier in cardiovascular research. Our current classification of MMPs based on few substrates is an oversimplification of a complex area of biology. This study provides a more comprehensive assessment of potential MMP substrates in the vasculature and represents a valuable resource for future investigations.
Human umbilical vein endothelial cells are the most widely used in vitro model for endothelial cells. Their secreted proteins, however, have not been comprehensively analysed so far. In this study, we accomplished to map the secretome of human umbilical vein endothelial cells by combining free-flow electrophoresis with nanoflow LC-MS/MS. This comprehensive analysis provides a basis for future comparative studies of protein secretion by endothelial cells in response to cardiovascular risk factors and is available on our website http://www.vascular-proteomics.com.
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