The Power of LC-MS Based Multiomics: Exploring Adipogenic Differentiation of Human Mesenchymal Stem/Stromal Cells

Evelyn Rampler, Dominik Egger, Harald Schoeny, Mate Rusz, Maria Pires Pacheco, Giada Marino, Cornelia Kasper, Thomas Naegele, Gunda Koellensperger

The molecular study of fat cell development in the human body is essential for our understanding of obesity and related diseases. Mesenchymal stem/stromal cells (MSC) are the ideal source to study fat formation as they are the progenitors of adipocytes. In this work, we used human MSCs, received from surgery waste, and differentiated them into fat adipocytes. The combination of several layers of information coming from lipidomics, metabolomics and proteomics enabled network analysis of the biochemical pathways in adipogenesis. Simultaneous analysis of metabolites, lipids, and proteins in cell culture is challenging due to the compound's chemical difference, so most studies involve separate analysis with unimolecular strategies. In this study, we employed a multimolecular approach using a two-phase extraction to monitor the crosstalk between lipid metabolism and protein-based signaling in a single sample (similar to 10(5) cells). We developed an innovative analytical workflow including standardization with in-house produced C-13 isotopically labeled compounds, hyphenated high-end mass spectrometry (high-resolution Orbitrap MS), and chromatography (HILIC, RP) for simultaneous untargeted screening and targeted quantification. Metabolite and lipid concentrations ranged over three to four orders of magnitude and were detected down to the low fmol (absolute on column) level. Biological validation and data interpretation of the multiomics workflow was performed based on proteomics network reconstruction, metabolic modelling (MetaboAnalyst 4.0), and pathway analysis (OmicsNet). Comparing MSCs and adipocytes, we observed significant regulation of different metabolites and lipids such as triglycerides, gangliosides, and carnitine with 113 fully reprogrammed pathways. The observed changes are in accordance with literature findings dealing with adipogenic differentiation of MSC. These results are a proof of principle for the power of multimolecular extraction combined with orthogonal LC-MS assays and network construction. Considering the analytical and biological validation performed in this study, we conclude that the proposed multiomics workflow is ideally suited for comprehensive follow-up studies on adipogenesis and is fit for purpose for different applications with a high potential to understand the complex pathophysiology of diseases.

Department of Analytical Chemistry, Department of Inorganic Chemistry, Mass Spectrometry Centre, Department of Microbiology and Ecosystem Science
External organisation(s)
Universität für Bodenkultur Wien, Ludwig-Maximilians-Universität München
No. of pages
Publication date
Peer reviewed
Austrian Fields of Science 2012
104002 Analytical chemistry
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