HTC-15 - Abstract

Abstract Information :

Lipidomics approaches are nowadays widely adopted to have a comprehensive view on lipid profiles in biological samples. Alterations in lipid profiles may provide a panel of biomarkers for diagnostic and prognostic purposes of various diseases. Untargeted lipidomics workflows may generate new hypothesis for support biological interpretation or complement other omics data. Targeted lipidomics approaches are often utilized to validate biomarkers found in a discovery phase or measure particular lipids associated with a certain disease or present in low abundance (e.g. lipid mediators in inflammation). In untargeted lipidomics, UHPLC-MS/MS with data-dependent acquisition (DDA) using Orbitrap or QTOF instruments is state of art. In DDA, subsequent to an MS full scan (survey scan) a certain number of MS/MS experiments are performed in which the most intensive precursor ions from the survey scan are selected for fragmentation to obtain MS/MS spectra for identification. This acquisition mode has some disadvantages including limited reproducibility of MS/MS experiments and incomplete coverage by MS/MS data. For this reason, we investigate the performance and advantages of UHPLC-ESI-QTOF-MS/MS with data independent acquisition (DIA) workflows using SWATH (sequential window acquisition of all theoretical fragment ion mass spectra). In SWATH, precursor ions for simultaneous fragmentation are selected with intermediate Q1 mass window width (e.g. typically 10-30 u) and the generated fragments readout by TOF analysis creating composite MS/MS spectra for each SWATH window. This leads to comprehensive MS and MS/MS data so that XICs can be generated both from precursors and fragments. This allows to select the most suitable signal (precursor or fragment ions) that shows the best sensitivity or better selectivity for generation of XICs and data processing, respectively. MS/MS spectra are available for identification over the entire chromatogram and all samples improving lipid coverage and identification percentage. On the other hand, DIA with SWATH can also provide better performance for quantitative analysis. In targeted lipidomics, triple-quadrupole instruments are the method of choice an data acquisition is done by SRM. This provides a good quantitation performance, yet only preselected targets are detected. On the other hand, if SWATH is used for quantitative analysis, besides the targets, the sensitivity and assay specificity of which can be improved by using narrow precursor isolation windows, also other lipids are simultaneously detected in the FULL MS scan and MS/MS data of other lipids are available from the other SWATH windows. This allows combination of targeted analysis with similar performance as QqQ with untargeted profiling. Possibilities, advantages and challenges of DIA with SWATH in untargeted and targeted lipidomics workflows will be discussed on clinical examples. In one study, lipid extracts of platelets of patients with stable angina pectoris and acute coronary syndrome have been profiled against controls and revealed significant alterations in lipid profiles of platelets which correlated with disease severity to some extent. In another study, the advantages of SWATH acquisition for targeted analysis of steroid hormones in plasma samples of patients treated with steroid hormone patches will be discussed.