Computational protocol: Pharmacometabolomic Signature of Ataxia SCA1 Mouse Model and Lithium Effects

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Protocol publication

[…] A Gerstel MPS2 automatic liner exchange system was used to inject 0.5 µl of sample at 50°C (ramped to 250°C) in splitless mode with 25 seconds splitless time. An Agilent 6890 gas chromatograph (Santa Clara CA) was used with a 30 m long, 0.25 mm i.d. Rtx5Sil-MS column with 0.25 µm 5% diphenyl film and an additional 10 m integrated guard column (Restek, Bellefonte PA). Chromatography was performed at a constant flow of 1 ml/minute, ramping the oven temperature from 50°C to 330°C with 22 minutes total run time. Mass spectrometry was performed using a Leco Pegasus IV time-of-flight mass spectrometer with a 280°C transfer line temperature, electron ionization at −70 V and an ion source temperature of 250°C. Mass spectra were acquired from m/z 85–500 at 17 spectra s-1 and 1850 V detector voltage.Result files were exported to the Fiehnlab servers and further processed by the in-house metabolomics BinBase database. All database entries in BinBase were matched against the Fiehn mass spectral library of 1,200 authentic metabolite spectra using retention index and mass spectrum information or the National Institute of Standards and Technology, version 5 commercial library. Identified metabolites were reported if they were present in at least 50% of the samples per study design group (as defined in the SetupX database). Quantitative data were normalized to the sum intensities of all known metabolites and used for statistical investigation. [...] Multivariate statistics and one-way analysis of variance (ANOVA) models were calculated using Statistica data miner V8 (Statsoft, Tulsa OK, USA). The ANOVA results were used for box-whisker plots and tables, and were subsequently converted into a Cytoscape node attribute file which was utilized to visualize the differential statistics on network graphs. Multivariate partial least square analysis was performed using the non-linear iterative partial lease squares algorithm using unit standard deviation scaling, 50 iterations with 0.0001 as convergence criterion and 7-fold cross validation of the resulting partial least square (PLS) models. […]

Pipeline specifications

Software tools BinBase, SetupX, Statistica
Applications Miscellaneous, MS-based untargeted metabolomics
Organisms Mus musculus
Diseases Machado-Joseph Disease
Chemicals Amino Acids, Glucose, Lithium, Purines