Integrated use of LC/MS/MS and LC/Q-TOF/MS targeted metabolomics with automated label-free microscopy for quantification of purine metabolites in cultured mammalian cells

Abstract

Purine metabolites have been implicated as clinically relevant biomarkers of worsening or improving Parkinson’s disease (PD) progression. However, the identification of purine molecules as biomarkers in PD has largely been determined using non-targeted metabolomics analysis. The primary goal of this study was to develop an economical targeted metabolomics approach for the routine detection of purine molecules in biological samples. Specifically, this project utilized LC/MS/MS and LC/QTOF/MS to accurately quantify levels of six purine molecules in samples from cultured N2a murine neuroblastoma cells. The targeted metabolomics workflow was integrated with automated label-free digital microscopy, which enabled normalization of purine concentration per unit cell in the absence of fluorescent dyes. The established method offered significantly enhanced selectivity compared to previously published procedures. In addition, this study demonstrates that a simple, quantitative targeted metabolomics approach can be developed to identify and quantify purine metabolites in biological samples. We envision that this method could be broadly applicable to quantification of purine metabolites from other complex biological samples, such as cerebrospinal fluid or blood.

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Abbreviations

ADP:

Adenosine diphosphate

AMP:

Adenosine monophosphate

ANOVA:

Analysis of variance

ATP:

Adenosine triphosphate

CNS:

Central nervous system

CSF:

Cerebrospinal fluid

DI-ESI-MS:

Direct-infusion electrospray ionization mass spectrometry

DL:

Detection limit

DMEM:

Dulbecco’s Modified Eagle Medium

ESI:

Electrospray ionization

FBS:

Fetal bovine serum

G6PD:

Glucose-6-phosphate-dehydrogenase

GC-MS:

Gas chromatography-mass spectrometry

HRMS:

High-resolution mass spectrometry

ICH:

International Conference on Harmonization

LC/MS/MS:

Liquid chromatography-tandem mass spectrometry

LC/QTOF/MS:

Liquid chromatography-quadrupole-time-of-flight-mass spectrometry

L-DOPA:

L-3,4-dihydroxyphenylalanine

LLOD:

Lower limit of detection

MRM:

Multiple reaction monitoring

N2a:

Neuro-2a

NADPH:

Nicotinamide adenine dinucleotide phosphate

NMR:

Nuclear magnetic resonance

NTPDase:

Ectonucleoside triphosphate diphosphohydrolase

PD:

Parkinson’s disease

QL:

Quantification limit

QTOF/MS:

Quadrupole-time-of-flight-mass spectrometry

UPDRS:

Unified Parkinson’s Disease Rating Scale

UPLC:

Ultra performance liquid chromatography

UV-vis:

Ultraviolet-visible spectroscopy

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Acknowledgements

LC/MS/MS targeted metabolomics experiments were performed at the Michigan State University Mass Spectrometry and Metabolomics Core Facility headed by Professor Dr. Dan Jones. The authors thank Dr. Anthony Schilmiller and Mrs. Lijun Chen for their help in conducting mass spectrometry analysis.

Funding

This study was funded by a Ferris State University Exceptional Merit Grant to S.E.N. and an American Association of Colleges of Pharmacy New Investigator Award to J.T.L.

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All authors contributed to all aspects of experimentation and manuscript preparation.

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Correspondence to Jennifer T. Lamberts.

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S. Eric Nybo declares that he has no conflict of interest.

Jennifer T. Lamberts declares that she has no conflict of interest.

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Nybo, S.E., Lamberts, J.T. Integrated use of LC/MS/MS and LC/Q-TOF/MS targeted metabolomics with automated label-free microscopy for quantification of purine metabolites in cultured mammalian cells. Purinergic Signalling 15, 17–25 (2019). https://doi.org/10.1007/s11302-018-9643-2

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Keywords

  • Purine
  • Label-free microscopy
  • Cell culture
  • Mass spectrometry
  • Metabolomics