Chromatof version 4

Muscle samples were stored in liquid nitrogen. Powder was prepared and a mixture of methanol-chloroform-water (MCW) (5:2:1/v:v:v) (Methanol LC-MS-grade, Chloroform Reagent Plus 99,8% Sigma-Aldrich) with cinnamic acid as internal standard (Sigma-Aldrich) was added. Samples were shaken at 750 rpm and 4 °C for 60 min. After addition of water (half volume), samples were centrifuged for 10 min at 5000 g to separate the polar (top), lipid (bottom) and interface (tissue debris) layers. The polar phase was dried under vacuum for 12 h. Metabolite analysis was performed on a gas chromatography coupled to time of flight mass spectrometer (Pegasus III- TOF-MS-System, LECO Corp., St. Joseph, MI, USA), complemented with an auto-sampler (MultiPurpose Sampler 2 XL, Gerstel, Mülheim an der Ruhr, Germany) as described in^{60 (link)}. Data analysis was performed using ChromaTOF Version 4.42 (LECO). The Golm metabolome database (GMD) was used to identify substances with respect to spectra-similarity and retention index. Data matrices for relative quantification were extracted from the mass spectra using MetMax software^{61 (link)}. Data were normalized to cinnamic acid for further analysis.

The vendor software ChromaTOF Version 4.42 (LECO) was used for metabolite evaluation with the following parameters: baseline offset of 1, peak width of 4 s, signal/noise of 20, and peak smoothing of 11 data points. Retention indices were calculated based on retention index standards. The Golm Metabolome Database (GMD) provided mass spectra and retention information for peak identification
[20 (link)]. The quantification routine of ChromaTOF was used for external calibration based on the measured quantification standards. Exported .txt files included the following: name, quantification mass retention index, first dimension retention time, second dimension retention time, area, concentration, match, reverse, quantitative signal/noise, type, concentration units, and the peak true spectrum in absolute values. For further data analysis, the tool MetMax was developed in cooperation with the MPIMP in Potsdam-Golm (http://gmd.mpimp-golm.mpg.de/apps/metmax)
[21 (link)]. MetMax provided the extraction of peak areas and quantities (retention analysis mode) and intensities of pre-defined mass ranges (isotope concentrator mode) from the exported .txt files. The in-house-developed pSIRM-wizard enabled the determination of ¹³C-label incorporation based on the exported data following the descriptions and equations stated in the paper. The R package is available on request.