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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

  • S. Eric Nybo
  • Jennifer T. LambertsEmail author
Original Article
  • 42 Downloads

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.

Keywords

Purine Label-free microscopy Cell culture Mass spectrometry Metabolomics 

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

Notes

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.

Author contributions

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

Funding information

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.

Compliance with ethical standards

Conflict of interest

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

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

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

11302_2018_9643_MOESM1_ESM.docx (36.4 mb)
ESM 1 (DOCX 37293 kb)

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.College of Pharmacy, Department of Pharmaceutical SciencesFerris State UniversityBig RapidsUSA

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