Rapid estimation of the energy charge from cell lysates using matrix-assisted laser desorption/ionization mass spectrometry: Role of in-source fragmentation

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 acetylcoenzyme 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-TRAP). By adding C-13-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 [C-13]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. (C) 2013 Elsevier Inc. All rights reserved.