Abstract
Negative ion mode nano-ESI-MS is often considered for the analysis of acidic compounds, including nucleotides. However, under high aqueous separation conditions, corona discharge is frequently observed at emitter tips, which may result in low ion abundances and reduced nano-ESI needle emitter lifetimes. In this work, we introduce a sheathless CE-MS method for the highly efficient and sensitive analysis of nucleotides employing ESI in positive ion mode, thereby fully circumventing corona discharge. By using a background electrolyte of 16mM ammonium acetate (pH 9.7) a mixture of 12 nucleotides, composed of mono-, di-, and tri-phosphates, could be efficiently analyzed with plate numbers per meter above 220000 and with LODs in the range from 0.06 to 1.3nM, corresponding to 0.4 to 8.6 attomole, when using an injection volume of about 6.5 nL only. The utility of the method was demonstrated for the profiling of nucleotides in low numbers of mammalian cells using HepG2 cells as a model system. Endogenous nucleotides could be efficiently analyzed in extracts from 50000 down to 500 HepG2 cells only. Moreover, apart from nucleotides, also some nicotinamide-adenine dinucleotides and amino acids could be analyzed under these conditions, thereby clearly illustrating the utility of this approach for metabolic profiling of low amounts of biological material.
Highlights
In metabolomics, LC hyphenated to high-resolution accurate mass TOF-MS is routinely used for discovery studies
When compared to the state-of-the-art ionpair nanoscale RP-anion-exchange LC–MS (LC–MS) method developed for profiling nucleotides and other anionic metabolites [23], which provided absolute LOD values of 100, 250, and 750 amol for adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP), respectively, our method showed an improvement in LOD of 19, 104, and 326-fold, respectively, for these compounds
We have developed a sheathless CZE–MS method for the highly sensitive and efficient profiling of nucleotides in low numbers of mammalian cells
Summary
LC hyphenated to high-resolution accurate mass TOF-MS is routinely used for discovery studies. The highly efficient profiling of polar and charged metabolites remains a challenge with modern LC columns including HILIC and ion-exchange LC, especially for phosphorylated metabolites such as nucleotides [1], which play key Abbreviations: ADP, adenosine diphosphate; AEC, adenylate energy charge; AMP, adenosine monophosphate; ATP, adenosine triphosphate; cAMP, cyclic AMP; DMEM F-12, DMEM/Nutrient Mixture F-12 Ham; ISVF, ionspray voltage floating; LLE, liquid–liquid extraction; RMT, relative migration times; t-ITP, transient-isotachophoresis roles in cell signaling and metabolism. A highly efficient microscale separation technique coupled to a high end MS instrument is required in order to enable the analysis of nucleotides in low amounts of biological material
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