Improved phosphometabolome profiling applying isotope dilution strategy and capillary ion chromatography-tandem mass spectrometry

Abstract

The phosphometabolome is comprised of all phosphorylated metabolites including the major metabolite classes sugar phosphates and nucleoside phosphates. Phosphometabolites are invaluable in any cell as a part of primary- and energy- metabolism, and as building blocks in the biosynthesis of macromolecules. Here, we report quantitative profiling of the phosphometabolome by applying capillary ion chromatography-tandem mass spectrometry (capIC-MS/MS), ensuring improved chromatographic separation, robustness and quantitative precision. Baseline separation was achieved for six out of eight tested hexose phosphates. Quantitative precision and reproducibility was improved by introducing a fully uniformly (U) C-labeled biological extract and applying an isotope dilution (ID) correction strategy. AC-labeled biological extract does in principle contain internal standards (IS) for all metabolites, but low abundant metabolites pose a challenge, and solutions to this are discussed. The extreme reproducibility and reliability of this capIC-MS/MS method was demonstrated by running the instrumentation continuously for ten days.

PCNA has specific functions in regulation of metabolism in haematological cells

“…All cell suspensions were immediately quenched in LN2, and extracted, lyophilized and cleared of protein as described in (Kvitvang & Bruheim, 2015), before reconstitution in compatible solvents for downstream analysis. TCA intermediates and phosphorylated metabolites were prepared for and quantified by capIC-MS/MS as described in (Kvitvang et al, 2014), with the modifications described in (Stafsnes, Rost, & Bruheim, 2018) employed for the haematological cells. Amino acids in DU145 and Hek293 extracts were quantified by GC-MS/MS as described in (Kvitvang, Andreassen, Adam, Villas-Boas, & Bruheim, 2011).…”

Absolute Quantification of the Central Carbon Metabolome in Eight Commonly Applied Prokaryotic and Eukaryotic Model Systems

“…Phosphorylated metabolites and intermediates of the TCA cycle were quantified by capillary ion chromatography (capIC)-MS/MS. Metabolite extracts were processed and analyzed as described in [39] with the modifications described in [40], employing a Xevo TQ-XS triple quadrupole mass spectrometer (Waters, Milford, MA, USA).…”

“…Metabolite extracts were added 20% (v/v) of an U 13 C-labeled S. cerevisiae extract prepared as described in [40] supplemented with U 13 C-pyruvic acid and -lactic acid (2440-0.5 and 1579-0.5, Cambridge isotope laboratories, Tewksbury, MA, USA), and derivatized as described in [70] to allow for quantification of organic acids. LC-MS/MS analysis was performed on an ACQUITY I-Class UPLC coupled to a Xevo TQ-XS triple quadrupole mass spectrometer (Waters) equipped with an electrospray source operating in positive mode.…”

“…Only a few labs apply Ion Chromatography (IC) on a routine basis, likely due to low throughput and high-maintenance instrumentation. Yet, it has superior resolution of phosphorylated metabolites [39][40][41]. For hydrophobic analytes such as long-chain fatty acids and lipids, supercritical fluid chromatography (SFC) is particularly well suited.…”

Metabolic Profiling of Glucose-Fed Metabolically Active Resting Zymomonas mobilis Strains

“…The measurement of phosphorylated sugars and nucleotides were performed using capillary ion chromatography [40,41] coupled to tandem mass spectrometry Xevo TQ-XS Triple Quadrupole Mass Spectrometry (Waters, Milford, MA, USA). Free amino acids and Organic acids were derivatized by Edman's reagent [42] and O-Benzylhydroxylamine [43], respectively.…”