A Comprehensive Workflow for Routine, Automated, Metabolite + Lipid
Analysis of Mammalian Cells
1. Introduction

Genevieve C. Van de Bittner, Mark Sartain, Alex Apffel, Thu T.A. Nguyen,Kristin B. Bernick, Manuel Gomez, Dustin Chang, Brian P. Smart,Reid Brennen, Christine Miller, Steven Fischer, Laurakay Bruhn
Agilent Technologies, Inc., Santa Clara, CA, USA

Metabolomics
2020
#74

2. Results and Discussion - Lipids

We developed an automatable workflow that harnesses a novel room temperature cell lysis and Dual Metabolite + Lipid Workflow Provides Similar Lipid Extraction Profiles to a Liquid-Liquid
quenching method and, from the same sample, extracts and separates polar metabolites and lipids Extraction Workflow
using solid phase extraction (SPE). To reduce hands-on time, increase throughput, and increase
robustness of the workflow, the majority of workflow steps were automated on a liquid handling
platform. This automated workflow was utilized to analyze the metabolic perturbations in K562
leukemia cells following treatment with methotrexate (MTX), which halts cellular proliferation.
Technological Advancements Provide a Room Temperature Dual Metabolite + Lipid Cell Sample
Preparation Workflow
Figure 1. Heat map of 6 extraction replicates from a liquid-liquid extraction workflow and the dual
The new dual metabolite + lipid cell sample prep workflow is easily automated due to a room temperature
quenching step and solid phase separation of polar metabolites and lipids.

Methotrexate Alters Lipid Abundances for Select Lipid Classes
Lipid class analysis of cells treated with MTX shows both increases and decreases in abundance for different lipidclasses. Of note, the class decrease in phosphatidylinositols (PIs) is dominated by decreases in short-chain PIs.

metabolite + lipid workflow showing 207 lipid annotations that represent 21 lipid classes. Lipidsignatures are consistent between the two workflows.

Untargeted Lipidomics Analysis finds a New Result

Dual Metabolite + Lipid Sample Prep Workflow Applied to a Model System
Application of the dual metabolite + lipid workflow to investigation of MTX treatment of K562 cells led tonew biological insights.

An untargeted metabolomics study1of MTX pharmacological activity in acell model found decreases andincreases in lipid classes and specificlipid species. Our comprehensive cell
workflow additionally found anincrease in acylcarnitines upon MTX
treatment, a finding not previously
reported1.
Figure 2. Heat map showing
unsupervised clustering of lipids
found in vehicle- and MTX- treatedsamples using the dual sample
prep workflow and Lipid Annotator,
Profinder, and MPP analysis.

3. Results and Discussion – Polar Metabolites
Dual Metabolite + Lipid Workflow Provides Excellent Polar Metabolite Recoveries when
Compared to a Liquid Extraction Workflow

Methotrexate Alters Polar Metabolite Abundances

A recent targeted metabolomics approach2 found MTX treatment led to accumulation of the purine metabolite intermediates
AICA riboside (AICAr) / AICAR resulting in activation of AMPK. This led to increased bioenergetic capacity and decreased onecarbon metabolism and cellular proliferation. The AICAr / AICAR findings were confirmed in this study.

Figure 3. Methotrexate-induced lipidomic signature in K562 leukemia cells using the comprehensive dual metabolite + lipid cellsample prep and data analysis workflow. As previously shown by Funk et al.1, ceramides (Cer_NS) increase with MTX treatment
and phosphatidylinositols (PI) decrease with MTX treatment, particularly shorter PIs.

4. Conclusions
We describe a new room temperature, automated cell sample prep workflow that provides effective
room temperature quenching of metabolism and enables extraction and separation of metabolites
and lipids from the same sample using solid phase extraction. We demonstrated excellent polar
metabolite recoveries and lipid signatures comparable to a liquid-liquid extraction method. Thissample prep workflow, coupled with Lipid Annotator, Profinder, and Mass Profiler Professional data
analysis packages, provides a comprehensive sample prep and data analysis workflow for analysis of
metabolomic and lipidomic perturbations in cells. Using this workflow for an analysis of the impact of
MTX on K562 cells revealed similar changes in metabolomic and lipidomic profiles as in prior studies
and uncovered a previously unreported increase in acylcarnitines. This method development andvalidation lays the groundwork for routine, automated, intra-sample multi-omics analysis of
mammalian cell samples.

References
1Funk, R.S. et al. Metabolomic Profiling to Identify Molecular Biomarkers of Cellular Response to Methotrexate In Vitro.

Figure 4. Recoveries from pre- and post-spiked 13C-labeled metabolites.

Figure 5. Methotrexate-induced polar metabolite signature in K562 leukemia cells (left), volcano plot indicating metabolites that increaseor decrease significantly upon MTX treatment (middle), and AICAr / AICAR peaks (right) in vehicle and MTX-treated metabolite extracts.
Note: due to the pandemic, a historical MS1 dataset from K562 cells prepared using a liquid extraction workflow was utilized.

Clin

Transl Sci. 2020 January; 13(1): 137-146.
2Papadopoli, D.J. et al. Methotrexate elicits pro-respiratory and anti-growth effects by promoting AMPK signaling. Scientific
Reports. 2020 May 12; 10(1): 7838.
For Research Use Only. Not for use in diagnostic procedures.
RA.0392824074