Bulletin of the Korean Chemical Society

Korean Chemical Society (대한화학회)
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Non-enzymatic Self-acetylation of α-Cyclosophorotridecaoses Isolated from Ralstonia solanacearum: Mass Spectrometric Study

  • Cho, Eunae (Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center (BMIC) & Institute for Ubiquitous Information Technology and Applications (CBRU), Konkuk University) ;
  • Lee, Sanghoo (Department of Bioanalysis, Seoul Medical Science Institute) ;
  • Jung, Seunho (Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center (BMIC) & Institute for Ubiquitous Information Technology and Applications (CBRU), Konkuk University)
  • Received : 2014.04.29
  • Accepted : 2014.05.15
  • Published : 2014.08.20
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Abstract

Keywords

Experimental

Bacterial Strains and Culture Conditions. R. solanacearum KACC 10698 was grown in a TGY medium at 24 ℃ with agitation. S. meliloti was cultured in a GMS medium to late logarithmic phase at 30 ℃.

Preparation of α-C13. α-C13 produced by R. solanacearum KACC 10698 was purified as described in our previous report.14 The acetylated and unsubstituted forms of α-C13 were treated with 0.1 M KOH at 37 ℃ for 1 h. The alkaline-treated materials were neutralized and desalted on a Bio-Gel P-4 column. The final unsubstituted α-C13 was confirmed with NMR spectroscopy and MALDI-TOF MS.

Preparatin of L13 from α-C13. L13 were prepared by acid hydrolysis (0.08 M trifluoroacetic acid, 100 ℃, 1 h) and semi-preparative high-performance liquid chromatography (HPLC). After the hydrolysis, the hydrolysates were evaporated to dryness to remove trifluoroacetic acid, and desalted by using a Sephadex G-10 column.25 The desalted material was subjected to semi-preparative HPLC (Agilent Technologies 1200 series) on a C18 column (5 µm, 250 × 9.4 mm; Eclipse XDB-C18) at 15 ℃, and detected with an RI detector using 99:1 (v/v) water:methanol as the solvent system at a flow rate of 1 mL/min. The product was analyzed with MALDI-TOF MS.

Purification of Cys from S. meliloti. The isolation and purification of Cys were carried out as previously described.26

Reaction Condition. Reactions were performed in a minimal reaction buffer containing 300 mM NaCl, 5 mM MnCl2, and 20 mM Tris-chloride buffer (pH 8.3) at 30 ℃ for 1 h.27 The concentrations of both substrates, coenzyme A (acetyl and succinyl), and α-C13 were 500 µM. After 1 h, the reaction mixture was lyophilized and the mass was detected with MALDI-TOF MS.

MALDI-TOF MS Analysis. The reaction mixture was first analyzed with MALDI-TOF MS using 2,5-dihydroxybenzoic acid as the matrix with a MALDI-TOF mass spectrometer (Voyager-DETM STR BioSpectrometry; Per-Septive Biosystems; Framingham, MA, USA) in positive ion mode.

ESI MS/MS Analysis. For detailed structural analysis, the ESI MS/MS technique was used. The reaction mixtures described above were desalted with Sephadex G-10 and lyophilized. The materials were dissolved in a 1:1 solution of water and MeOH and directly infused into the ESI source at a rate of 1 mL/min. The low-energy CAD experiments were carried out on an API 4000TM triple quadrupole LC/ MS/MS system (Applied Biosystems; Foster City, CA, USA) equipped with a turbo ESI source. Ionization was performed in positive ion mode and nitrogen was used as the drying and nebulizing gas. The spray voltage was set at 4500 eV in product ion scan mode (MS2) and the scan range was m/z 1600 to 2500. The applied CEs were 50, 55, and 70 eV.

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