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
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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.
KeywordsPurine Label-free microscopy Cell culture Mass spectrometry Metabolomics
Analysis of variance
Central nervous system
Direct-infusion electrospray ionization mass spectrometry
Dulbecco’s Modified Eagle Medium
Fetal bovine serum
Gas chromatography-mass spectrometry
High-resolution mass spectrometry
International Conference on Harmonization
Liquid chromatography-tandem mass spectrometry
Liquid chromatography-quadrupole-time-of-flight-mass spectrometry
Lower limit of detection
Multiple reaction monitoring
Nicotinamide adenine dinucleotide phosphate
Nuclear magnetic resonance
Ectonucleoside triphosphate diphosphohydrolase
Unified Parkinson’s Disease Rating Scale
Ultra performance liquid chromatography
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.
All authors contributed to all aspects of experimentation and manuscript preparation.
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.
This article does not contain any studies with human participants or animals performed by any of the authors.
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