ALBERT

Description: Glycosyl inositol phospho ceramides (GIPCs) are the major sphingolipids on earth, as they account for a considerable fraction of the total lipids in plants and fungi, which in turn represent a large portion of the biomass on earth. Despite their obvious importance, GIPC analysis remains challenging due to the lack of commercial standards and automated annotation software. In this work, we introduce a novel GIPC glycolipidomics workflow based on reversed-phase ultra-high pressure liquid chromatography coupled to high-resolution mass spectrometry. For the first time, automated GIPC assignment was performed using the open-source software Lipid Data Analyzer (LDA), based on platform-independent decision rules. Four different plant samples (salad, spinach, raspberry, and strawberry) were analyzed and the results revealed 64 GIPCs based on accurate mass, characteristic MS2 fragments and matching retention times. Relative quantification using lactosyl ceramide for internal standardization revealed GIPC t18:1/h24:0 as the most abundant species in all plants. Depending on the plant sample, GIPCs contained mainly amine, N-acetylamine or hydroxyl residues. Most GIPCs revealed a Hex-HexA-IPC core and contained a ceramide part with a trihydroxylated t18:0 or a t18:1 long chain base and hydroxylated fatty acid chains ranging from 16 to 26 carbon atoms in length (h16:0–h26:0). Interestingly, four GIPCs containing t18:2 were observed in the raspberry sample, which was not reported so far. The presented workflow supports the characterization of different plant samples by automatic GIPC assignment, potentially leading to the identification of new GIPCs. For the first time, automated high-throughput profiling of these complex glycolipids is possible by liquid chromatography-high-resolution tandem mass spectrometry and subsequent automated glycolipid annotation based on decision rules.

Description: In the highly dynamic field of metabolomics, we have developed a method for the analysis of hydrophilic metabolites in various biological samples. Therefore, we used hydrophilic interaction chromatography (HILIC) for separation, combined with a high-resolution mass spectrometer (MS) with the aim of separating and analyzing a wide range of compounds. We used 41 reference standards with different chemical properties to develop an optimal chromatographic separation. MS analysis was performed with a set of pooled biological samples human cerebrospinal fluid (CSF), and human plasma. The raw data was processed in a first step with Compound Discoverer 3.1 (CD), a software tool for untargeted metabolomics with the aim to create a list of unknown compounds. In a second step, we combined the results obtained with our internally analyzed reference standard list to process the data along with the Lipid Data Analyzer 2.6 (LDA), a software tool for a targeted approach. In order to demonstrate the advantages of this combined target-list based and untargeted approach, we not only compared the relative standard deviation (%RSD) of the technical replicas of pooled plasma samples (n = 5) and pooled CSF samples (n = 3) with the results from CD, but also with XCMS Online, a well-known software tool for untargeted metabolomics studies. As a result of this study we could demonstrate with our HILIC-MS method that all standards could be either separated by chromatography, including isobaric leucine and isoleucine or with MS by different mass. We also showed that this combined approach benefits from improved precision compared to well-known metabolomics software tools such as CD and XCMS online. Within the pooled plasma samples processed by LDA 68% of the detected compounds had a %RSD of less than 25%, compared to CD and XCMS online (57% and 55%). The improvements of precision in the pooled CSF samples were even more pronounced, 83% had a %RSD of less than 25% compared to CD and XCMS online (28% and 8% compounds detected). Particularly for low concentration samples, this method showed a more precise peak area integration with its 3D algorithm and with the benefits of the LDAs graphical user interface for fast and easy manual curation of peak integration. The here-described method has the advantage that manual curation for larger batch measurements remains minimal due to the target list containing the information obtained by an untargeted approach.