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

Jordan Wilkins, Dhananjay Sakrikar, Xuan Mai Petterson, Ian R Lanza, Eugenia D Trushina

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article number83
JournalMetabolomics
Volume15
Issue number6
DOIs
StatePublished - Jun 1 2019

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Metabolomics
Fibroblasts
Metabolites
Skin
Mass Spectrometry
Citric Acid Cycle
Mass spectrometry
Liquid chromatography
Methanol
Amino Acids
Workflow
Recovery
Metabolic Networks and Pathways
Liquid Chromatography
Gas Chromatography
Disease Progression
Gas chromatography
Cells
Throughput
acylcarnitine

Keywords

  • Acylcarnitines
  • Amino acids
  • Cell harvesting
  • Metabolomics
  • Skin fibroblasts
  • TCA cycle

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Biochemistry
  • Clinical Biochemistry

Cite this

A comprehensive protocol for multiplatform metabolomics analysis in patient-derived skin fibroblasts. / Wilkins, Jordan; Sakrikar, Dhananjay; Petterson, Xuan Mai; Lanza, Ian R; Trushina, Eugenia D.

In: Metabolomics, Vol. 15, No. 6, 83, 01.06.2019.

Research output: Contribution to journalArticle

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