Pcdl database

Tissue samples (50–70 mg) were homogenized using 20 volumes of cold methanol as previously described [14 (link)]. Metabolite extracts were subjected to mass spectrometry using an HPLC 1290 series coupled to an ESI-Q-TOF MS/MS 6520 (Agilent Technologies, Santa Clara, CA, USA) as previously described [14 (link)]. Multivariate statistic analyses were performed using Metaboanalyst platform [19 ]. The preliminary identification of differential metabolites (Student’s T-Test, Benjamini Hochberg False Discovery Rate, p<0.05) was performed using the PCDL database from Agilent (Agilent Technologies, Barcelona, Spain), which accounts retention times in a standardized chromatographic system, exact mass and isotope distribution as an orthogonal searchable parameters to complement accurate mass data (AMRT approach) according to previously published works [14 (link)]. The version of the PCDL database used had retention times and accurate mass data for 679 compounds. To complete the identification process we searched for unidentified metabolites in Metlin Database (https://metlin.scripps.edu/index.php) which includes accurate masses and MS/MS spectrum for 961.829 molecules.

Metabolites were extracted from plasma samples of 8 random samples from cohort 1 with methanol according to a previously described method36 (link). Briefly, 30 μl of cold methanol was added to 90 μl of plasma, incubated 1 h at −20 °C and centrifuged for 3 min at 12000 g. The supernatants were recovered, evaporated using a Speed Vac (Thermo Fisher Scientific, Barcelona, Spain) and resuspended in water. We used an ultra-high pressure liquid chromatography (Agilent 1290 LC) system coupled to an electrospray-ionization quadrupole time of flight mass spectrometer (Q-TOF) 6520 instrument (Agilent Technologies, Barcelona, Spain). A column with 1.8 micron particle size was employed, and we performed identification of metabolites using the PCDL database from Agilent (Agilent Technologies, Barcelona, Spain), which uses retention times in a standardized chromatographic system as an orthogonal searchable parameter to complement accurate mass data, according to previously published works37 (link).

Metabolomics and lipidomics will be performed after the extraction of metabolites and lipids from plasma/serum at the baseline and final stages of the experiments. Metabolites and lipids will be separated by UPLC and detected by QTOF and TQP, depending on the procedure. Species identification will be performed via the PCDL database of Agilent Technologies, which uses retention times in a standardized chromatographic system as an orthogonal searchable parameter to complement accurate mass data (accurate mass retention time approach). In both cases, the SMPDB database [38 (link), 39 (link)] will be used for querying metabolites obtained from the HMDB search, using a 0.05 Da M.W. tolerance and the Metaboanalyst platform [40 , 41 ] for the determination of differential features.