title: Rapid estimation of the energy charge from cell lysates using matrix-assisted laser desorption/ionization mass spectrometry: role of in-source fragmentation.
abstract: Nucleotides are key players in the central energy metabolism of cells. Here we show how to estimate the energy charge from cell lysates by direct negative ion matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using 9-aminoacridine as matrix. We found a high level of in-source decay of all the phosphorylated nucleotides, with some of them producing considerable amounts of adenosine-5'-diphosphate (ADP) fragment ions. We investigated the behavior of adenosine-5'-monophosphate (AMP), ADP, and adenosine-5'-triphosphate (ATP) as well as the cofactors coenzyme A (CoA) and acetyl-coenzyme A (ACoA) and nicotinamide adenine dinucleotides (NAD⁺ and NADH) in detail. In-source decay of these compounds depends strongly on the applied laser power and on the extraction pulse delay. At standard instrument settings, the 9-aminoacridine (9-AA) matrix resulted in a much higher in-source decay compared with 2,4,6-trihydroxyacetophenone (2,4,6-THAP). By adding ¹³C-labeled ATP to a cell lysate, we were able to determine the degree of in-source decay during an experiment. Analyzing a cell extract of the monocytic cell line THP-1 with [¹³C]ATP as internal standard, we were able to obtain values for the energy charge that were similar to those determined by a reference liquid chromatography electrospray ionization coupled to mass spectrometry (LC-ESI-MS) method.
authors.affiliation: Department of Chemistry and Applied Biosciences, Eidgenössiche Technische Hochschule Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Robert F
authors.initials: RF
authors.lastname: Steinhoff
authors.affiliation: Department of Chemistry and Applied Biosciences, Eidgenössiche Technische Hochschule Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Jasmin
authors.initials: J
authors.lastname: Krismer
authors.affiliation: Department of Chemistry and Applied Biosciences, Eidgenössiche Technische Hochschule Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Klaus
authors.initials: K
authors.lastname: Eyer
authors.affiliation: Department of Chemistry and Applied Biosciences, Eidgenössiche Technische Hochschule Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Stephan R
authors.initials: SR
authors.lastname: Fagerer
authors.affiliation: Department of Chemistry and Applied Biosciences, Eidgenössiche Technische Hochschule Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Ibàñez
authors.affiliation: Department of Chemistry and Applied Biosciences, Eidgenössiche Technische Hochschule Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Pabst
authors.affiliation: Department of Chemistry and Applied Biosciences, Eidgenössiche Technische Hochschule Zurich, CH-8093 Zurich, Switzerland. Electronic address: zenobi@org.chem.ethz.ch.
authors.forename: Renato
authors.initials: R
authors.lastname: Zenobi
mesh_terms: D000098:Acetophenones / Q000737:chemistry; D000585:Aminacrine / Q000737:chemistry; D002457:Cell Extracts; D004734:Energy Metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013997:Time Factors
chemical_list: D000098:Acetophenones; D002457:Cell Extracts; D000585:Aminacrine; C107067:2,4,6-trihydroxyacetophenone
keywords: ATP; Energy charge; In-source decay; MALDI–MS; Metabolite analysis; Nucleoside phosphates