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A comprehensive protocol for multiplatform metabolomics analysis in patient-derived skin fibroblasts

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Abstract

Introduction

Patient-derived skin fibroblasts offer a unique translational model to study molecular mechanisms of multiple human diseases. Metabolomics profiling allows to track changes in a broad range of metabolites and interconnected metabolic pathways that could inform on molecular mechanisms involved in disease development and progression, and on the efficacy of therapeutic interventions. Therefore, it is important to establish standardized protocols for metabolomics analysis in human skin fibroblasts for rigorous and reliable metabolic assessment.

Objectives

We aimed to develop an optimized protocol for concurrent measure of the concentration of amino acids, acylcarnitines, and components of the tricarboxylic acid (TCA) cycle in human skin fibroblasts using gas (GC) and liquid chromatography (LC) coupled with mass spectrometry (MS).

Methods

The suitability of four different methods of cell harvesting on the recovery of amino acids, acylcarnitines, and TCA cycle metabolites was established using GC/MS and LC/MS analytical platforms. For each method, metabolite stability was determined after 48 h, 2 weeks and 1 month of storage at − 80 °C.

Results

Harvesting cells in 80% methanol solution allowed the best recovery and preservation of metabolites. Storage of samples in 80% methanol up to 1  month at − 80 °C did not significantly impact metabolite concentrations.

Conclusion

We developed a robust workflow for metabolomics analysis in human skin fibroblasts suitable for a high-throughput multiplatform analysis. This method allows a direct side-by-side comparison of metabolic changes in samples collected at different time that could be used for studies in large patient cohorts.

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Data availability

All data generated or analyzed during this study are included in this publication. All raw data collected in this study was uploaded to the Metabolomics Workbench: NIH Data Repository (website: https://www.metabolomicsworkbench.org/).

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Acknowledgements

This study was supported by the National Institutes of Health NIEHS, NIA, NINDS (Grant Numbers R01ES020715, RF1AG 55549, R01NS107265) and Mayo Clinic Metabolomics Core (NIDDK Grant Number U24DK100469); Mayo Clinic Clinomics; and Mayo Clinic Center for Multiple Sclerosis and Autoimmune Neurology

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Authors and Affiliations

  1. Department of Neurology, Mayo Clinic, Rochester, MN, 55905, USA

    Jordan Wilkins & Eugenia Trushina

  2. Metabolomics Core Laboratory, Mayo Clinic, Rochester, MN, 55905, USA

    Dhananjay Sakrikar, Xuan-Mai Petterson & Ian R. Lanza

  3. Division of Endocrinology, Mayo Clinic, Rochester, MN, 55905, USA

    Ian R. Lanza

  4. Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, 200 First Street SW, Guggenheim Bld., Room 1542B, Rochester, MN, 55905, USA

    Eugenia Trushina

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  1. Jordan Wilkins
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  2. Dhananjay Sakrikar
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  3. Xuan-Mai Petterson
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  4. Ian R. Lanza
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  5. Eugenia Trushina
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Contributions

JW, XP, DS, IL, and ET contributed to the design of this study, data analysis and interpretation. JW conducted the experiments and performed the majority of the data analysis. DS and XP conducted metabolomics profiling. JW and ET wrote the manuscript. All authors edited the manuscript and approved the final version.

Corresponding author

Correspondence to Eugenia Trushina.

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All authors declare no conflict of interest.

Ethical approval

All ethical guidelines and procedures established by the Mayo Clinic Institutional Review Board for the use of human skin fibroblasts were followed in this study.

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Wilkins, J., Sakrikar, D., Petterson, XM. et al. A comprehensive protocol for multiplatform metabolomics analysis in patient-derived skin fibroblasts. Metabolomics 15, 83 (2019). https://doi.org/10.1007/s11306-019-1544-z

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