Quantitation of intracellular purine intermediates in different Corynebacteria using electrospray LC-MS/MS
- PDF / 333,828 Bytes
- 11 Pages / 595.276 x 790.866 pts Page_size
- 47 Downloads / 148 Views
ORIGINAL PAPER
Quantitation of intracellular purine intermediates in different Corynebacteria using electrospray LC-MS/MS Susanne Peifer & Konstantin Schneider & Gudrun Nürenberg & Dietrich A. Volmer & Elmar Heinzle
Received: 30 July 2012 / Revised: 23 August 2012 / Accepted: 23 August 2012 # Springer-Verlag 2012
Abstract Intermediates of the purine biosynthesis pathway play key roles in cellular metabolism including nucleic acid synthesis and signal mediation. In addition, they are also of major interest to the biotechnological industry as several intermediates either possess flavor-enhancing characteristics or are applied in medical therapy. In this study, we have developed an analytical method for quantitation of 12 intermediates from the purine biosynthesis pathway including important nucleotides and their corresponding nucleosides and nucleobases. The approach comprised a single-step acidic extraction/quenching procedure, followed by quantitative electrospray LC-MS/MS analysis. The assay was validated in terms of accuracy, precision, reproducibility, and applicability for complex biological matrices. The method was subsequently applied for determination of free intracellular pool sizes of purine biosynthetic pathway intermediates in the two Gram-positive bacteria Corynebacterium glutamicum and Corynebacterium ammoniagenes. Importantly, no ion pair reagents were applied in this approach as usually required for liquid chromatography analysis of large classes of diverse metabolites. Keywords Biological samples . HPLC . Mass spectrometry . Nucleic acid sampling . Quantitative extraction . Bioanalytical methods S. Peifer : K. Schneider : E. Heinzle (*) Biochemical Engineering Institute, Saarland University, Campus A1.5, 66123 Saarbrücken, Germany e-mail: [email protected] G. Nürenberg : D. A. Volmer Institute of Bioanalytical Chemistry, Saarland University, Campus B 2.2, 66123 Saarbrücken, Germany
Introduction Nucleotides and their corresponding nucleosides and nucleobases are essential biomolecules and fulfill various functions in the living cell. In addition to being central building blocks for DNA and RNA [1], they also act as co-substrates in enzymatic reactions, play crucial roles in activating metabolites, and serve as signal mediators [2]. Furthermore, several intermediates of the purine biosynthesis pathway exhibit additional characteristics—ranging from flavor-enhancing qualities (inosine monophosphate (IMP) and guanosine monophosphate (GMP) [3, 4]) to drug-assisted therapy (e.g., inosine and purine analogues [5, 6]). These properties make them attractive targets for the pharmaceutical and biotechnological industries. Particularly the fermentative production using Corynebacteria (e.g., Corynebacterium ammoniagenes) has been successfully performed over the past decades [7–10]. As a result, quantifying these substances from biological matrices such as bacteria is crucial to assess the potential of the underlying cellular systems. The quantitative determination of these compounds, however, is v
Data Loading...
