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MB Sample ID: SA008955
Local Sample ID: | 49 |
Subject ID: | SU000184 |
Subject Type: | Animal |
Subject Species: | Rattus norvegicus |
Taxonomy ID: | 10116 |
Genotype Strain: | SpragueDawley |
Gender: | female |
Species Group: | Mammal |
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Sample Preparation:
Sampleprep ID: | SP000184 |
Sampleprep Summary: | An i.v. bolus dose of [ring-13C6]phenylalanine (15 ?g g?1 body weight) was given 15 min prior to the animals being killed. Immediately after death, the gastrocnemius muscle was removed from the left and right hindlimb and split into a medial and lateral deep red and superficial white portion and frozen in liquid propane chilled by liquid nitrogen. Tissue fluid and mixed gastrocnemius muscle proteins were isolated according to the method of Ljungqvist et al. (1997). The mixed muscle protein precipitate from the isolation was hydrolysed by heating with 6 m HCl overnight at 110°C. Both tissue fluid and hydrolysed mixed gastrocnemius muscle amino acids were purified using a BioRad AG-50 × 8 ion exchange resin prior to mass spectrometry analysis. Tissue fluid The level of enrichment of [ring-13C6]phenylalanine in tissue fluid was analysed using ThermoFisher Quantum gas chromatography tandem mass spectrometry (GC/MS/MS) (San Jose, CA, USA). The heptafluorobutyryl isobutyl ester derivative was prepared as described by Ford et al. (1985) and the amino acids were measured under negative ion chemical ionisation conditions using isobutane as reactant gas. The [M-HF]? fragments reflecting the m0 and m+6 species were monitored (m/z transitions 397?377 and 403?383, respectively) and the enrichment of the label was measured against a calibration curve prepared from known amounts of labelled and unlabelled phenylalanine (range 030%). |