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Determination of online quenching efficiency for an automated cellular microfluidic metabolomic platform using mass spectrometry based ATP degradation analysis

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Abstract

As microfluidic cell culture progresses, the need for robust and reproducible intracellular analyses grows. In particular, intracellular metabolites are subject to perturbation and degradation during the lysing process. The reliability of intracellular metabolomic analysis in microfluidic devices depends on the preservation of metabolite integrity during sample preparation and storage. Described here is a novel automated microfluidic system exhibiting the necessary rapid cellular lysis and quenching of enzymatic activity. Quenching efficiency was assessed using a novel ratiometric MALDI-MS-based assay of exogenous isotopic adenosine triphosphate (ATP) hydrolysis to isotopic adenosine diphosphate (ADP) as a marker of metabolite degradation. The lysis system of the microfluidic device was enhanced using a Peltier cooler to chill the lysate and quench aberrant enzymatic activity. Parameter optimization (flow rate, collection time, and temperature control) improved the endogenous and exogenous ADP/ATP ratios by 44.9% and 39.8% respectively consistent with traditional quenching techniques. The effects of chilling/quenching on metabolism were evaluated resulting in over 500 significant features compared to non-chilled from untargeted capillary LC-MS metabolomic analyses. These include increased levels of tryptophan, histidine, and pyruvate as well as decreased levels in UDP-N-acetylglucosamine. The results illustrate the need for both rapid lysis and quenching in microfluidic cell culture platforms.

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Abbreviations

ATP:

Adenosine triphosphate

ADP:

Adenosine diphosphate

AMP:

Adenosine monophosphate

EC:

Endothelial cell

DMEM:

Dulbecco’s modified eagle medium

NAD+:

Nicotinamide adenine dinucleotide

UDP:

Uridine diphosphate

MeOH:

Methanol

ACN:

Acetonitrile

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Acknowledgments

The authors wish to thank Dr. Dave Wood of the Protein Core Facility in the Saint Louis University Department of Biochemistry for his assistance with MALDI analyses.

Funding

This work is supported by the National Institutes of Health (USA): 1R15GM113153 (JLE).

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Author notes

  1. Laura A. Filla and Katherine L. Sanders contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Saint Louis University, 3501 Laclede Ave, Saint Louis, MO, 63103, USA

    Laura A. Filla, Katherine L. Sanders, John B. Coulton, Robert T. Filla & James L. Edwards

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  1. Laura A. Filla
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  2. Katherine L. Sanders
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  3. John B. Coulton
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Correspondence to James L. Edwards.

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Filla, L.A., Sanders, K.L., Coulton, J.B. et al. Determination of online quenching efficiency for an automated cellular microfluidic metabolomic platform using mass spectrometry based ATP degradation analysis. Anal Bioanal Chem 411, 6399–6407 (2019). https://doi.org/10.1007/s00216-019-02018-3

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