An Automated, Combined Workflow for Extracting PolarMetabolites and Lipids from Mammalian Cells

Genevieve C. Van de Bittner1, Alex Apffel1, Thu T.A. Nguyen1, Kristin B. Bernick1, ManuelGomez2, Dustin Chang1, Mark Sartain2, Brian P. Smart1, Reid Brennen1, Christine Miller3,
Steven Fischer3, Laurakay Bruhn1

ASMS 2020
MP391

Agilent Research Labs1, Mass Spectrometry Division2, & Segment Marketing3 Agilent
Technologies, Inc., Santa Clara, CA, USA

Introduction

Results and Discussion

Cell Sample Preparation is Difficult, Time-Consuming, and Error-Prone
Challenges in Mammalian Cell Sample Preparation• Cold liquids (i.e. liquid nitrogen) used for fast metabolic quenching are difficult
and, in some cases, dangerous to handle, especially in a time-sensitive workflow• Liquid-liquid phase separations for metabolite and lipid separation require
visualization of the liquid-liquid interface for accurate phase separation
Technological Solutions for Mammalian Cell Sample Preparation• Room temperature 50% trifluoroethanol for cell lysis and metabolism quenching• Solid phase extraction (SPE) for sequential metabolite and lipid collection fromindividual samples• Automation of SPE workflow on Bravo Liquid Handler

Novel Workflow Provides Good Polar Metabolite Recoveries and Lipid Extraction that is Comparable to a
Traditional Workflow

Results and Discussion
13C Glutamine Stable Isotope Tracing Confirms Room Temperature Metabolism

QuenchingProteins precipitated from K562 cell lysate and soluble lipids are captured by the Captiva EMR-Lipid plate2, providing
a metabolite-containing flow-through. Lipids are sequentially eluted. Excellent metabolite recoveries for organic acid,amino acid, sugar, and nucleotide classes were measured from spiked-in 13C-labeled metabolites using ion-pairing
reversed-phase 6545 LC/Q-TOF and Quant 10.1 analysis. Representative lipid extractions analyzed by negative-ionmode, reversed-phase 6545 LC/Q-TOF, Lipid Annotator 1.0 and MPP 15.1 are presented in a heat map of relative peakareas for 225 lipid annotations representing 21 lipid classes across four extraction replicates from each workflow.

Conclusions

References

Improved, Semi-Automated Cell Sample Preparation
Technologies Application Note.
• Effective room temperature metabolism quenching
2Zhao, L. et. al. (2017) Agilent
improves safety and reduces complexity of workflow
No detectable conversion of 13C glutamine into 13C glutamate by K562 cellular enzymes
Technologies Application Note.
during cold temperature metabolism quenching or during room temperature• Unique Captiva EMR-Lipid SPE material enables sequential
metabolism quenching with 50% trifluoroethanol (TFE, v/v). ATP is stable at roomcollection of polar metabolites and lipids for intra-sample
3Matyash, V., et al., J Lipid Res 49,
temperature in 50% TFE for at least 4 hours and the amount of ATP extracted ismulti-omics correlations
1137-1146, (2008) doi:comparable to traditional cold quench workflows (not shown). Data analysis with
10.1194/jlr.D700041-JLR200• SPE workflow is compatible with Bravo automation, which
MassHunter VistaFlux Software.
can reduce operator errors and improve reproducibility
For Research Use Only. Not for use in diagnostic procedures.
1Hartman, T. E., et. al. (2017) Agilent