Targeted Mass Spectrometric Metabolite Profiling for Studies of Central Carbon Metabolism
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Metabolism is the total set of enzymatic reactions occurring in a cell, tissue, organ, or organism to sustain life. It is an implementation of all upstream levels of regulation and environmental stimuli and hence reflecting a biological system on the functional level. This leaves measurements of the small-molecule intermediates and products of metabolism, the metabolites, valuable to all areas of biology. Metabolite analyses increasingly rely on hyphenated mass spectrometric (MS) techniques providing unmatched sensitivity and specificity, and in particular, on liquid chromatography (LC)-MS offering broad coverage of the physicochemically diverse metabolite pool. Out of the total pool, about a hundred evolutionary conserved metabolites carry the majority of metabolic flux through a set of reactions referred to as central carbon metabolism. The fast cellular turnover of these metabolites and their high reactivity challenges accurate and precise quantification by LC-MS. Adding to this is the competitive nature of electrospray ionization, by which analytes gain charge before entering the mass analyzer. This renders the ionization efficiency of target metabolites highly dependent on the sample matrix constituents. This thesis presents targeted approaches compensating the inherent challenges of MS-based metabolite profiling to allow accurate quantitative analysis of central carbon metabolism in cellular model systems. First, the precision of quantitative analysis of the phosphometabolome, which comprises a significant share of central carbon metabolites, was increased by introducing isotope dilution. This strategy exploits stable isotopically labeled analogs of all metabolites to compensate degradation and matrix-dependent ionization efficiency. These analogs are not commercially available but were prepared from a reference organism cultivated on an isotopically labeled substrate. Next, zwitterionic hydrophobic interaction liquid chromatography (HILIC) was exploited to chromatographically resolve the polar pyridine nucleotides NAD and NADP. Combined with a tailored extraction procedure, this allowed for rapid and highly sensitive MS analysis of these metabolites operating at the center of cellular redox homeostasis. Compensation strategies beyond isotope dilution were strictly required for accurate quantification and hence, for calculation of cellular redox ratios. Further, phosphometabolome profiling was combined with parallel quantitative LC-MS analyses covering the other major constituents of central carbon metabolism, namely organic- and amino acids, to report the intracellular concentration of 68 central carbon metabolites in eight commonly applied prokaryotic and eukaryotic biological model systems. Such comprehensive datasets are scarce even in databases dedicated to reporting the total set of metabolites, the metabolome, of various organisms. Finally, integration of the quantitative metabolite profiling approach with complementary transcriptome and proteome profiling approaches substantiated non-canonical roles of the cellular scaffold protein proliferating cell nuclear antigen (PCNA). Impairment of PCNA interactions occurring through the conserved interaction motif AlkB homologue 2 PCNA-interacting motif (APIM) led to a sharp reduction in intermediates of the glycolytic and pentose phosphate pathway of central carbon metabolism, and in the high-energy carrying nucleoside phosphates in haematological cancer cells. This did not occur in cells from solid tissue nor primary monocytes from healthy donors. Extrinsic stress did, however, provoke the same response in primary monocytes, suggesting stress as a modulator of this response, which was later reproduced in a larger panel of newly established multiple myeloma cell lines. Integration of central carbon metabolome and kinase enriched proteome profiles substantiated PCNA as a central regulator of cellular processes beyond its established roles in DNA replication and repair in the nucleus. More specifically, it suggested PCNA as a regulator of cytoplasmic stress through regulation of central carbon metabolism in haematological cells. The five studies of this thesis present and apply state of the art MS approaches for accurate quantitative analysis of central carbon metabolism. Together, they demonstrate how comprehensive and accurate reporting of these high-flux metabolites can be applied to advance our understanding of biological model systems and their inner workings.