Lipid Atlas of Keratinocytes and Betulin Effects on its Lipidome Profiled by Comprehensive UHPLC–MS/MS with Data Independent Acquisition Using Targeted Data Processing

Abstract

Betulin is a pentacyclic triterpene with demonstrated healing properties in mid-dermal wounds. A few earlier studies have provided insights into the wound healing effects on the molecular level. However, there are still questions left on the molecular targets of betulin. Therefore, a pharmacolipidomics analysis of betulin is undertaken in human immortalized keratinocytes to monitor alterations in the lipid profiles induced by treatment with betulin. For this purpose, lipid extracts of keratinocytes treated with betulin and untreated controls are comprehensively analyzed by an untargeted UHPLC-ESI-QTOF-MS/MS lipidomics profiling workflow using data-independent acquisition. Targeted data processing allows the identification of 611 lipid species from 21 different lipid classes. Statistical analysis of the identified lipids shows significant changes in 440 lipid species that can be described as downregulation of cholesteryl esters and triacylglycerides and upregulation of glycerophospholipids, sphingolipids, and diacylglycerides. Additionally, some other signals corresponding to triterpenes are found in the betulin group and suggested that betulin is incorporated (in the membrane) and metabolized in keratinocytes.

Platelet ACKR3/CXCR7 favors antiplatelet lipids over an atherothrombotic lipidome and regulates thromboinflammation

“…Untargeted (UHPLC-ESI-QTOF-MS/MS) and targeted (micro-UHPLC-ESI-QTrap-MS/MS) lipidomics analyses for oxylipins 43,44 were performed for resting and thrombin-activated platelets 45 and platelet supernatant/releasates 46 in presence/absence of CXCR7 agonist/vehicle control. Lipid extraction from platelet releasate was done with MTBE/MeOH/H 2 O; from platelet pellet in a monophasic extraction with isopropanol/water 9:1 (vol/vol), followed by solid-phase extraction on Bond Elut Certify II cartridges (Agilent) with ethyl acetate/n-hexane/acetic acid 75:24:1 (vol/vol/v) for oxylipins.…”

Gentiana lutea Extract Modulates Ceramide Synthesis in Primary and Psoriasis-Like Keratinocytes

“…Specifically, the level of the ceramides NS and NdS was increased [36]. Very recently, it has been shown by the lipidomics profiling of human immortalized keratinocytes that the pentacyclic triterpene betulin differentially regulates the content of 440 lipid species, including increased ceramides [37].…”

Recent methodological developments in data-dependent analysis and data-independent analysis workflows for exhaustive lipidome coverage

“…SWATH experiments were performed to characterize 214 and 140 lipids from immortalized keratinocytes in both positive and negative ionization modes, respectively. As statistical treatment of the data is mainly based on retention times, authors strongly advise to add internal standards to correct any deviation (Calderón et al, 2020;Cebo et al, 2021). Interestingly, SWATH can be employed for compound annotation using MS/MS library search but also quantification at the MS/MS level because, unlike classic DDA, it offers continuous MS/MS spectra acquisition for particular fragment ions.…”