MSACL 2022 Abstract

Introduction:
Nucleotide biosynthesis is crucial for DNA synthesis and cellular function. Quantitative determination of nucleosides and nucleotides in cells is fundamental to understanding nucleotide biosynthesis, energy metabolism and signalling pathways in different diseases. Currently available mass spectrometry-based methods have low compound coverage, e.g., less than 10 nucleosides/nucleotides, or a high rate of false identifications in the analysis of biological samples. Ultrahigh-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) can provide confident qualification with structural information of fragment ions when combined with appropriate internal standards. The objective of this study was to develop a robust method to simultaneously quantify nucleotide metabolites from both de novo and salvage biosynthesis pathways with increased molecular coverage and high sensitivity for biomedical studies.

Method:
Intracellular nucleotides/nucleosides were extracted by a two-step protein precipitation method after including selected stable-isotope labelled internal standards. To cover more intermediates from the studied pathways, chromatographic separation was carried out on two LC columns. A Waters ACQUITY UPLC™ BEH amide (1.7 μm 100 × 2.1 mm) column was chosen for nucleobases and nucleosides, while a Thermo HyperCarb™ porous graphitic carbon (PGC, 3 μm 50 x 2.1 mm) column was used to monitor nucleotides. Parallel reaction monitoring (PRM) MS was used to measure a total of 28 nucleotide metabolites against internal calibrators of authentic standards. Quality controls were prepared by spiking different levels of known amounts of analytes in water and extracting with the same protocol. This method was applied to the analysis of reprogramming of nucleotide synthesis in Mll-Af9 knock-in acute myeloid leukemia (AML) mouse model cells.

Results:
PRM with HRMS has unique advantages over popular MRM in permitting identifications with high confidence identification while eliminating co-eluting interferences of compounds with the same MRM transitions. Furthermore, it provides the flexibility to choose a combination of characteristic fragmentation ions for quantification. ESI spectra of nucleotides and nucleosides give abundant fragmentation with ions derived from three structural subunits, i.e. nucleobase, ribose, and phosphate (or their combinations). The main fragmentation ions include, nucleobase-derived (for bases A, G, C, T, U, and Hx at m/z 134.046, 150.041, 110.035, 125.034, 111.019, and 135.030 respectively, and the products of their fragmentation), phosphate-derived (H2PO4- at m/z 96.968 and H2PO4--H2O at m/z 78.957), and phosphoribose-derived (m/z 211.001 and 195.006 for phosphoribo- and phosphodeoxyribo- analytes, respectively). With two analytical columns, and a total run time, i.e. 30 min, a larger coverage and baseline separation of historically challenging analytes including ATP and dGTP (identical molecular weight with same MRM transition and about 1000 fold cellular concentration difference) was achievable. This method was validated on linearity, sensitivity, accuracy, and reproducibility. The potential of this methodology was illustrated in studies of AML mouse model cell lines on a gene mutation with high frequencies and associates with adverse outcome in leukemia patients. Preliminary data revealed that mutation of this gene disrupts nucleotide biosynthesis homeostasis for unrestrained leukemia cell proliferation, an important finding since upregulated purine and pyrimidine synthesis serves to supply dNTPs for cancer cell growth.

Conclusion:
A comprehensive, validated and reliable MS-based method targeting nucleotide biosynthesis and metabolism was developed to profile and quantify 28 nucleotides, nucleosides and bases, applicable to biological and clinical matrices and with versatility to be expanded to include other nucleoside and nucleotide derivatives in metabolomics studies.