Tri sil htp

For the ¹³C isotopomer analysis of intracellular metabolites by GC-MS, snap-frozen NHAs and HCNs were thawed and centrifuged to remove the protein precipitations. Sodium 2-oxobutyrate (50 nmol) was added to the supernatants as an internal standard. The samples were evaporated and derivatized by trimethylsilylation (Tri-Sil HTP, Thermo Scientific). A total of 3 µL of the derivatized solution was injected onto an Agilent 6970 gas chromatograph equipped with a fused silica capillary GC column (30-m length/0.25-mm diameter) coupled with an Agilent 5973 mass selective detector [51 (link)]. The measured distributions of the carbon isotopomers were corrected for the natural abundance of the ¹³C isotopomer (1.1%).

EgP samples (2 mg) and monosaccharide standards were treated with the same procedure. First, 100 µL of the standard stock solution of 1 mg mL⁻¹ of each monosaccharide was dried under nitrogen gas flow. Second, the samples of polysaccharides, and a mixture containing the standard monosaccharides included in the Internal Standard (IS), were methanolyzed in 2 mL metanol/3 M HCl at 80 °C for 24 h. The monosaccharides, glucose, galactose, rhamnose, fructose, mannose, xylose, apiose, and myo-inositol (IS) (Sigma-Aldrich, St. Louis, MO, USA), as well as pyridine, hexane and metanol/3 M HCl solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). Then, the saccharides were washed with methanol and dried under nitrogen gas flow. Third, the Trimethylsilyl reaction was accomplished with 200 µL of Tri-Sil HTP (Thermo Fisher Scientific, Franklin, MA, USA). Each vial with the sample was heated at 80 °C for 1 h. The derived sample was cooled to room temperature and dried under a stream of nitrogen. Fourth, the dry residue was extracted with hexane (2 mL) (Sigma-Aldrich, St. Louis, MO, USA), and centrifuged. Finally, the hexane solution containing silylated monosaccharides was concentrated and reconstituted in hexane (200 µL), filtered and transferred to a GC-MS autosampler vial (Thermo Fisher Scientific, Franklin, MA, USA). Sample preparation and analyses were performed in triplicate.

The enzymatic reaction mixtures were lyophilized, and the products were extracted with MeOH. The MeOH extract was then dried and Tri-Sil HTP (Thermo Scientific, Waltham, MA) (100 μl) was added and left to stand for 20 min. The solvent was removed in a flow of argon gas, and the silylated products were extracted with hexanes (100 μl) and injected into the GC-MS (Hewlett Packard 5890 SERIES II Gas chromatograph).