Pooled WT or tn-/- L3 larvae (25 each) were selected and washed with NaCl/0.1% Triton to remove food or debris. Each batch of pooled larvae were flash frozen in liquid nitrogen and sent to the University of Utah Metabolomics Core. Metabolites were extracted and derivatized before gas chromatography-mass spectrometry (GC-MS) analysis with an Agilent 5977B GC-MS. Data was collected using MassHunter software and metabolite identity was determined using MassHunter Quant. The metabolite data was normalized to standards, parsed, and Metaboanalyst 3.0 was used for statistical analysis and data processing. Independently, groups of 10 larvae were weighed on an analytical scale to determine the difference in body mass between WT and mutant cohorts before analysis. Six biological replicates were performed for each genotype.
5977b gcms
Manufactured by Agilent Technologies
The 5977B GC/MS is an advanced gas chromatography-mass spectrometry system designed for high-performance analytical applications. It combines a gas chromatograph with a mass spectrometer to provide reliable separation, identification, and quantification of a wide range of chemical compounds. The system features a robust and sensitive design for consistent and reproducible results.
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3 protocols using 5977b gcms
1
Comparative Larval Metabolomics Analysis
2
Synthesis of Compounds 1-16
3
Synthesis of Compounds 1-16
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Chemicals for the synthesis of compounds 1–16 were acquired from commercial sources and used as received. Products were purified on Biotage® SNAP KP-Sil silica gel columns using an automated Isolera™ chromatography system with elution detection set at λ 254 nm. Mass spectra were recorded on an Agilent 5977B GCMS. 1H and 13C NMRs were recorded on a Bruker AVANCE™ III HD 300 MHz spectrometer and referenced to TMS. Bacillithiol (BSH) trifluoroacetic acid salt and monobromobimane were purchased from Biosynth Carbosynth (United Kingdom) and Cayman Chemical (Ann Arbour, MI), respectively.
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