M6P and GDP-mannose measure
Description
We measured the levels of M6P and GDP-mannose in frozen HCC tumor and adjacent normal tissues from patients (n=42 pairs). Notably, both metabolites were elevated in tumor tissues compared to their matched normal counterparts. All metabolite levels were normalized to the average levels observed in adjacent normal tissues.
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Steps to reproduce
Polar metabolite extraction was performed as previously described. Cells at 80% confluence or homogenized tissue samples were collected and extracted with 1 mL extraction solvent (50% methanol, 20% Milli-Q water, 30% acetonitrile) containing internal standards (13C-glutamine). After sonication and -20°C extraction for 30 min, samples were centrifuged (16,100g, 4°C, 10 min). Supernatants were freeze-dried, reconstituted in mobile phase, and stored at -80°C for LC-MS analysis. Protein concentration was determined by BCA assay. Targeted metabolomics analysis was performed using a TSQ Quantiva mass spectrometer (Thermo Scientific) in negative ion mode, coupled with an Ultimate 3000 UHPLC system (Thermo Scientific) equipped with a HILIC carbon column (Waters, 186004868). The mobile phases were buffer A (95% water, 5% acetonitrile, 20 mM ammonium acetate,20 mM ammonium hydroxide, pH 9.0) and buffer B (100% acetonitrile). The LC gradient was as follows: 0–3 min, 15% A; 3–16 min, gradient to 50% A; 16–20 min, gradient to 85% A; 20–23 min, return to 15% A; 23–25 min, hold at 15% A. Metabolites were quantified based on total ion count peak areas, normalized to internal standards and protein concentrations. Natural isotope correction was applied to account for isotopic contributions during quantification. Retention times were verified using standards within the same batch of samples, ensuring accurate identification and quantification.