Tri sil

Glycosyl composition analysis was performed at the Complex Carbohydrate Research Center (CCRC at University of Georgia) by combined gas chromatography-mass spectrometry (GC-MS) of the per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis as described previously [35 (link)]. Briefly, the dry sample (200 µg) was heated with methanolic HCl in a sealed screw-top glass test tube for 16 h at 80 °C. After cooling and removal of the solvent under a stream of nitrogen, the samples were re-N-acetylated and dried again. The sample was then derivatized with Tri-Sil^® (Thermoscientific, Waltham, MA, USA) at 80 °C for 30 min. GC-MS analysis of the TMS methyl glycosides was performed on an Agilent 7890A GC interfaced to a 5975C MSD, using an Supelco Equity-1 fused silica capillary column (30 m × 0.25 mm ID). The GC temperature program conditions were as follows: initial temperature of 80 °C with 2 min hold; ramp at 20 °C/min to 140 °C with 2 min hold; ramp at 2 °C/min to 200 °C; ramp at 30 °C/min to 250 °C with five minute hold.

Mice were fasted for 16 h before intraperitoneal injection of D[U-¹³C] glucose (Cambridge Isotopes; 2 g kg⁻¹ body mass) and analysed 30 min later. Tumours were surgically excised, immediately placed in ice-cold 50% methanol in watch and homogenized. Metabolites were extracted by three cycles of freezing in liquid nitrogen and thawing in a 37 °C water bath. Macromolecules and debris were removed by centrifugation and the supernatants containing soluble metabolites were dried and derivatized for 30 min at 42 °C in 100 μl of a trimethylsilyl donor (Tri-Sil, Thermo). Metabolites were analysed using an Agilent 6970 gas chromatograph networked to an Agilent 5973 mass selective detector. ¹³C enrichment analysis was performed as previously described63 (link).