Liquid Chromatography-Mass Spectrometry Metabolic and Lipidomic Sample Preparation Workflow for Suspension-Cultured Mammalian Cells using Jurkat T lymphocyte Cells

Abstract

Metabolomics is the comprehensive study of metabolism as it pertains to an organism or biological system. Lipidomics, a subset discipline of metabolomics, encompasses the study of cellular lipid functions: including pathways, networks, and interactions. The abundance of metabolites and lipids, along with their contribution to health and disease, makes metabolomics a valuable tool for biomarker research. Disease biomarker identification requires a reproducible, sensitive, and accurate analytical platform. Although transcriptomic and proteomic areas have well-established protocols for sample preparation and data processing, the metabolomics field is still developing comparable standardized conventions. Furthermore, of the few comparative LC-MS metabolomic studies that have been applied to mammalian cell cultures, most are targeted to adherent cell lines. The purpose of this work was to optimize a sample preparation workflow for the cellular metabolomic analysis of suspension-cultured mammalian cells using commercially available Jurkat T lymphocytes as a model system. The current investigation evaluated commonly used sample preparation techniques for reproducibility, accuracy, and applicability to untargeted biomarker discovery. Results show ammoniated cell rinsing solutions to be an effective means to remove extracellular components present in the media without causing ion suppression or affecting the integrity of the cellular membrane. Additionally, a novel workflow was designed to allow for the combined analysis of metabolites and lipids from mammalian suspension cells from a single cell pellet. The Folch lipid extraction protocol was coupled to an 80% MeOH metabolite isolation to ensure high extraction efficiency for phospholipids and triacylglycerides. While the workflow was tailored to cells in suspension, it could also be applied to adherent cell lines.

Optimization of Folch, Bligh-Dyer, and Matyash sample-to-extraction solvent ratios for human plasma-based lipidomics studies

“…Metabolite data processing and analysis was performed with XCMS R Script, Thermo Compound Discoverer, and MetaboAnalyst 3.0, respectively. Lipid data processing was performed using MZmine [27] to generate a feature list ( m/ z, retention time, and peak area). Peak detection was performed by mass detection, chromatogram smoothing, peak deconvolution, deisotoping, feature alignment, and gap-filling.…”

LipidPioneer : A Comprehensive User-Generated Exact Mass Template for Lipidomics

“…Lipids were extracted from the cells using the original Folch procedure [12] allowing for a chloroform:methanol:water ratio of 8:4:3 (v/v). Details outlining this procedure [13] can be found in the supplemental information.…”

Changes in the uterine metabolome of the cow during the first 7 days after estrus

“…All sodiated or potassiated adducts of the protonated ion and dimer or trimer complexes of the same ion were identified and removed from the data set. Metabolites were first identified by use of an in‐house curated retention time metabolite library (Ulmer, Yost, Chen, Mathews, & Garrett, ). Subsequently, additional external databases such as the human metabolome database (http://www.hmdb.ca) were used to expand the number of identified metabolites.…”