TY - JOUR

T1 - The Power of LC-MS Based Multiomics

T2 - Exploring Adipogenic Differentiation of Human Mesenchymal Stem/Stromal Cells

AU - Rampler, Evelyn

AU - Egger, Dominik

AU - Rusz, Mate

AU - Pacheco, Maria Pires

AU - Marino, Giada

AU - Kasper, Cornelia

AU - Schonen, Harald

AU - Nägele, Thomas

AU - Koellensperger, Gunda

N1 - Funding Information: Funding: The LILY project for the production of isotopically labeled lipid received funding from the Austrian BMWFW Federal Ministry (aws PRIZE prototype funding). Open Access Funding by the University of Vienna. Funding Information: Acknowledgments: This work is supported by the University of Vienna, the Faculty of Chemistry, the Vienna Metabolomics Center (VIME; http://metabolomics.univie.ac.at/) and the research platform Chemistry Meets

PY - 2019/10/8

Y1 - 2019/10/8

N2 - 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 (~105 cells). We developed an innovative analytical workflow including standardization with in-house produced 13C 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.

AB - 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 (~105 cells). We developed an innovative analytical workflow including standardization with in-house produced 13C 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.

KW - Fat differentiation

KW - LC-MS

KW - Lipidomics

KW - Mathematical modelling

KW - Mesenchymal stem cells

KW - Metabolomics

KW - Multiomics

KW - Network analysis

KW - Proteomics

KW - Stromal cells

UR - http://www.scopus.com/inward/record.url?scp=85073055884&partnerID=8YFLogxK

U2 - 10.3390/molecules24193615

DO - 10.3390/molecules24193615

M3 - Article

C2 - 31597247

AN - SCOPUS:85073055884

VL - 24

JO - MOLECULES

JF - MOLECULES

SN - 1420-3049

IS - 19

M1 - 3615

ER -