Metabolic adaptation to the host environment is a defining feature of the pathogenicity of Mycobacterium tuberculosis (Mtb), but we lack biochemical knowledge of its metabolic networks. Many bacteria use catabolite repression as a regulatory mechanism to maximize growth by consuming individual carbon substrates in a preferred sequence and growing with diauxic kinetics. Surprisingly, untargeted metabolite profiling of Mtb growing on ¹³C-labeled carbon substrates revealed that Mtb could catabolize multiple carbon sources simultaneously to achieve enhanced monophasic growth. Moreover, when co-catabolizing multiple carbon sources, Mtb differentially catabolized each carbon source through the glycolytic, pentose phosphate, and/or tricarboxylic acid pathways to distinct metabolic fates. This unusual topologic organization of bacterial intermediary metabolism has not been previously observed and may subserve the pathogenicity of Mtb.
A new interface procedure has been developed that allows, for the first time, the high-efficiency analysis of synthetic oligonucleotides up to 75 bases by reversed-phase HPLC and on-line electrospray ionization mass spectrometry. For oligonucleotides up to 30 bases in length, single-base resolution can be obtained with low levels of cation adduct formation in the negative ion electrospray mass spectra. A key part of the method uses 1,1,1,3,3,3-hexafluoro-2-propanol as an additive to the HPLC mobile phase, adjusted to pH 7.0 with triethylamine. This novel additive results in both good HPLC separation and efficient electrospray ionization. The broad potential of this new method is demonstrated for synthetic homopolymers of thymidine (PolyT), fragments based on the pBR322 plasmid sequence, and phosphorothioate ester antisense oligonucleotides. This approach will be of particular utility for the characterization of DNA probes and PCR primers and quality control of antisense compounds such as phosphorothioates and their metabolites, as well as of materials used in clinical trials.
While alterations in xenobiotic metabolism are considered causal in the development of bladder cancer (BCa), the precise mechanisms involved are poorly understood. In this study, we used high-throughput mass spectrometry to measure over 2,000 compounds in 58 clinical specimens, identifying 35 metabolites which exhibited significant changes in BCa. This metabolic signature distinguished both normal and benign bladder from BCa. Exploratory analyses of this metabolomic signature in urine showed promise in distinguishing BCa from controls, and also non-muscle from muscle-invasive BCa. Subsequent enrichment-based bioprocess mapping revealed alterations in phase I/II metabolism and suggested a possible role for DNA methylation in perturbing xenobiotic metabolism in BCa. In particular, we validated tumor-associated hypermethylation in the CYP1A1 and CYP1B1 promoters of BCa tissues by bisulfite sequence analysis and methylation-specific PCR, and also by in vitro treatment of T-24 BCa cell line with the DNA demethylating agent 5-aza-2′-deoxycytidine. Further, we showed that expression of CYP1A1 and CYP1B1 was reduced significantly in an independent cohort of BCa specimens compared to matched benign adjacent tissues. In summary, our findings identified candidate diagnostic and prognostic markers and highlighted mechanisms associated with the silencing of xenobiotic metabolism. The metabolomic signature we describe offers potential as a urinary biomarker for early detection and staging of BCa, highlighting the utility of evaluating metabolomic profiles of cancer to gain insights into bioprocesses perturbed during tumor development and progression.
Trifluoroacetic acid (TFA) and other volatile strong acids, used as modifiers in reverse-phase high-performance liquid chromatography, cause signal suppression for basic compounds when analyzed by electrospray ionization mass spectrometry (ESI-MS). Evidence is presented that signal suppression is caused by strong ion pairing between the TFA anion and the protonated sample cation of basic sample molecules. The ion-pairing process "masks" the protonated sample cations from the ESI-MS electric fields by rendering them "neutral. " Weakly basic molecules are not suppressed by this process. The TFA signal suppression effect is independent from the well-known spray problem that electrospray has with highly aqueous solutions that contain TFA. This previously reported spray problem is caused by the high conductivity and surface tension of aqueous TFA solutions. A practical method to enhance the signal for most basic analytes in the presence of signal-suppressing volatile strong acids has been developed. The method employs postcolumn addition of a solution of 75% propionic acid and 25% isopropanol in a ratio 1:2 to the column flow. Signal enhancement is typically 10-50 times for peptides and other small basic molecules. Thus, peptide maps that use ESI-MS for detection can be performed at lower levels, with conventional columns, without the need to use capillary chromatography or reduced mass spectral resolution to achieve satisfactory sensitivity. The method may be used with similar results for heptafluorobutyric acid and hydrochloric acid. A mechanism for TFA signal suppression and signal enhancement by the foregoing method, is proposed.
SUMMARY
Activity based metabolomic profiling (ABMP) allows unbiased discovery of enzymatic activities encoded by genes of unknown function. ABMP applies liquid chromatography-mass spectrometry (LC-MS) to analyze the impact of a recombinant enzyme on the homologous cellular extract as a physiologic library of potential substrates and products. The Mycobacterium tuberculosis protein Rv1248c was incompletely characterized as a thiamine diphosphate-dependent α-ketoglutarate decarboxylase. Here, recombinant Rv1248c catalyzed consumption of α–ketoglutarate in a mycobacterial small molecule extract with matched production of 5-hydroxylevulinate (HLA) in a reaction predicted to require glyoxylate. As confirmed using pure substrates by LC-MS, 1H-NMR, chemical trapping, and intracellular metabolite profiling, Rv1248c catalyzes C-C bond formation between the activated aldehyde of α–ketoglutarate and the carbonyl of glyoxylate to yield 2-hydroxy-3-oxoadipate (HOA), which decomposes to HLA. Thus, Rv1248c encodes a HOA synthase (HOAS).
Prostate cancer is the second leading cause of cancer related death in American men. Development and progression of clinically localized prostate cancer is highly dependent on androgen signaling. Metastatic tumors are initially responsive to anti-androgen therapy, however become resistant to this regimen upon progression. Genomic and proteomic studies have implicated a role for androgen in regulating metabolic processes in prostate cancer. However, there have been no metabolomic profiling studies conducted thus far that have examined androgen-regulated biochemical processes in prostate cancer. Here, we have used unbiased metabolomic profiling coupled with enrichment-based bioprocess mapping to obtain insights into the biochemical alterations mediated by androgen in prostate cancer cell lines. Our findings indicate that androgen exposure results in elevation of amino acid metabolism and alteration of methylation potential in prostate cancer cells. Further, metabolic phenotyping studies confirm higher flux through pathways associated with amino acid metabolism in prostate cancer cells treated with androgen. These findings provide insight into the potential biochemical processes regulated by androgen signaling in prostate cancer. Clinically, if validated, these pathways could be exploited to develop therapeutic strategies that supplement current androgen ablative treatments while the observed androgen-regulated metabolic signatures could be employed as biomarkers that presage the development of castrate-resistant prostate cancer.
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