Rapid estimation of the energy charge from cell lysates using matrix-assisted laser desorption/ionization mass spectrometry: Role of in-source fragmentation
Section snippets
Metabolites
ATP, ADP, AMP, flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma–Aldrich (Switzerland). β-Nicotinamide adenine dinucleotide (oxidized form) (NAD+), and β-nicotinamide adenine dinucleotide (oxidized form) disodium hydrate (NADH) were purchased from Acros Chemicals (Belgium). Acetyl-coenzyme A (ACoA) and coenzyme A (CoA) were purchased from AppliChem (Germany). All metabolites were used without further purification and were
In-source decay versus post-source decay
ATP was measured by MALDI–MS using 9-AA (90% EtOH) and, using standard instrument conditions, generated approximately 40% to 60% ADP and 5% AMP. Because reference measurements of the ATP standard by LC–MS revealed no hydrolysis products, we were confident that ATP was fragmented to ADP and AMP to a large extent during the MALDI–MS analysis. By repeating the measurement on the same spot in negative linear mode, a similar ratio among ATP, ADP, and AMP was acquired. Thus, we conclude that the
Conclusions
Adenosine-containing nucleotides and cofactors are present as highly abundant metabolites in all mammalian cells. Here we studied the in-source fragmentation behavior of ACoA, CoA, NADH, NAD+, and ATP to ADP and AMP in negative mode MALDI–MS and discussed how this influences the estimation of the energy charge. The degree of in-source fragmentation in ESI of adenosine phosphate molecules is low; using LC–ESI–MS, in-source fragmentation products are linked to their parent ions via the retention
Acknowledgments
We acknowledge funding from the European Union for the Marie Curie Initial Training Network ISOLATE (grant agreement 289995). We thank Dr. Sascha Sauer and Anja Freiwald from the Max-Planck-Institute for Molecular Genetics Berlin for the human monocytic cell line THP1. We also thank Louis Bertschi (MS service unit) for support with the LC measurements as well as Simon Weidmann and Christian Berchtold for helpful discussions.
References (41)
Mass spectrometric imaging of small molecules
Trends Biotechnol.
(2010)- et al.
In situ metabolomic mass spectrometry imaging: recent advances and difficulties
J. Proteomics
(2012) - et al.
Analytical strategies for LC–MS-based targeted metabolomics
J. Chromatogr. B
(2008) - et al.
Regulation of pathways of glucose metabolism in kidney
FEBS Lett.
(1982) - et al.
Single cell metabolomics
Curr. Opin. Biotechnol.
(2011) - et al.
MALDI–MS-based imaging of small molecules and proteins in tissues
Curr. Opin. Chem. Biol.
(2007) - et al.
Practical quantitative biomedical applications of MALDI–TOF mass spectrometry
Am. Soc. Mass Spectrom.
(2002) - et al.
Investigations of the metastable decay of DNA under ultraviolet matrix-assisted laser desorption/ionization conditions with post-source-decay analysis and hydrogen/deuterium exchange
Am. Soc. Mass Spectrom.
(1998) - et al.
Peptide and protein identification by matrix-assisted laser desorption ionization (MALDI) and MALDI–post-source decay time-of-flight mass spectrometry
Am. Soc. Mass Spectrom.
(1999) - et al.
Further studies of in-source fragmentation of peptides in matrix-assisted laser desorption–ionization
Int. J. Mass Spectrom. Ion Processes
(1997)
The energy charge of the adenylate pool as a regulatory parameter: interaction with feedback modifiers
Biochemistry
AMP-activated protein kinase: the energy charge hypothesis revisited
BioEssays
Metabolite measurements
Analysis of nucleosides and nucleotides in infant formula by liquid chromatography–tandem mass spectrometry
Anal. Bioanal. Chem.
Nucleotide and nucleotide sugar analysis by liquid chromatography–electrospray ionization–mass spectrometry on surface-conditioned porous graphitic carbon
Anal. Chem.
TagFinder for the quantitative analysis of gas chromatography––mass spectrometry (GC–MS)-based metabolite profiling experiments
Bioinformatics
13C NMR-based metabolomics for the classification of green coffee beans according to variety and origin
J. Agric. Food Chem.
Challenge of mass spectrometry toward the elucidation of life phenomena: molecular and functional analysis of cellular phenomena using single-cell mass spectrometry
Biol. Pharm. Bull.
Ultra-highly sensitive in situ metabolomic imaging for visualizing spatiotemporal metabolic behaviors
Anal. Chem.
Cited by (4)
MALDI-MS analysis and imaging of small molecule metabolites with 1,5-diaminonaphthalene (DAN)
2014, Journal of Mass SpectrometryQuantification of saquinavir from lysates of peripheral blood mononuclear cells using Microarrays and standard MALDI-TOF-MS
2014, Journal of the American Society for Mass Spectrometry