Sample preparation workflow for the liquid chromatography tandem mass spectrometry based analysis of nicotinamide adenine dinucleotide phosphate cofactors in yeast^†

Abstract

The accurate quantification of the highly unstable intracellular cofactor nicotinamide adenine dinucleotide phosphate in its oxidized and reduced forms demands a thorough evaluation of the analytical workflow and dedicated methods reflecting their solution chemistry as well as the biological importance of their ratio. In this work, we present a workflow for the analysis of intracellular levels of oxidized and reduced nicotinamide adenine dinucleotide phosphate in the yeast Pichia pastoris, including hot aqueous extraction, chromatographic separation in reversed-phase conditions employing a 100% wettable stationary phase, and subsequent tandem mass spectrometric analysis. A thorough evaluation and optimization of the sample preparation procedure resulted in excellent biological repeatabilities (on average <10%, N = 3) without employing an internal standardization approach. As a consequence, the methodology proved to be appropriate for the relative assessment of intracellular levels of oxidized and reduced nicotinamide adenine dinucleotide phosphate in different P. pastoris strains. The ratio of reduced versus oxidized nicotinamide adenine dinucleotide phosphate was significantly higher in an engineered strain overexpressing glucose-6-phosphate dehydrogenase than in the corresponding wildtype strain. Interestingly, a difference was also observed in the nicotinamide adenine dinucleotide phosphate pool size, which was significantly higher in the wildtype than in the modified strain.

Quantifying intracellular metabolites in yeast using a matrix with minimal interference from naturally occurring analytes

“…The reason for the high ratios was probably the oxidation of NADH and NADPH and, consequently, an increased concentration of NAD + and NADP + [32,33]. However, Hou and coworkers [39] reported data for the NAD + /NADH and NADP + /NADPH ratios that were close to those measured in the current study.…”

“…In the case of NADPH, only one peak for NADP + was observed. As previously reported in the literature [32], ammonium acetate (pH 8.0) has been shown to improve the stability of NADPH and reduce the oxidation. To investigate this, the standards of NADH and NADPH were prepared in 5 mM ammonium acetate (pH 8.0), which indeed reduced the oxidation of these two compounds.…”

Stable isotope labeling by essential nutrients in cell culture (SILEC) for accurate measurement of nicotinamide adenine dinucleotide metabolism

“…Ortmayr, et al , conducted an extensive stability and analysis validation on NAD(H)/NADP(H) and reported that NADH and NADPH have a relatively short half-life at a pH lower than 6, and NADPH was only stable at pH 8. 9 Therefore, for analysis of a large number of samples, buffering samples with 5 mM ammonium acetate at pH 8 may be desirable as the highly liable NADPH was stable for at least 24 hours under these conditions. LC-MS based analysis of NAD metabolites may also present some unique and unexpected challenges that analysts should be aware of.…”

“…A previous study by Ortmayr, et al specifically noted the lack of available stable isotope labeled analogs as an impediment to conducting rigorous and reproducible NAD metabolite analysis. 9 Furthermore, since LC-high resolution MS (LC-HRMS) also permits simultaneous metabolite quantitation and tracing with isotope-specific resolution, a diversity of stable isotope labels ( e.g. 2 H, 13 C, and/or 15 N) can be introduced by experimental design and distinguished by the HRMS.…”

Concentrations of Nicotinamide in Plasma by RP-HPLC With Fluorescence Detection

“…Our research shows that long-term nicotinamide overload can cause insulin resistance and oxidative stress, and eventually lead to diabetes [3,4] . Currently, the detection of nicotinamide in plasma samples is a complex method, which requires high instrument conditions and liquid chromatography -mass spectrometry [5][6][7] . This method is also highly timeconsuming, mainly due to the very low levels of nicotinamide in the plasma.…”