DOI: 10.25345/c5sk6s
title: MassIVE MSV000083353 - GNPS - "Pyramid" gradient benchmarking dataset (QTOF data only)
title: Pyramid gradient benchmarking dataset (QTOF only) Description: Generated by the Dorrestein lab, it consists of mixtures of 4 samples prepared at different sample ratios. Method: They were analysed on the Q-Exactive and the Maxis II (qTOF), in three chromatographic conditions (C18, C18RTshift, and C18polar). 
Material and methods. All solvents and reactants used were LC-MS grade. Samples. Four samples were used in the gradient benchmarking dataset: - The NIST 1950 SRM human serum sample. See the following manuscript for more informations (https://pubs.acs.org/doi/suppl/10.1021/ac402503m) - Two human fecal samples collected from a male individual with a 35 days interval (Sample F1= 11-10-2013, and F2= 12-14-2013). Samples are part of the American Gut Project (https://msystems.asm.org/content/3/3/e00031-18) - A tomato seedling sample (Solanum lycopersicum) was prepared using 3 weeks post germination specimens (fresh whole seedling was used). Sample preparation. The NIST SRM 1950 sample (1mL), the two fecal samples (210 mg of fresh material each), and the tomato seedling (800 mg of fresh material) were dissolved in 1 mL of 70/30 MeOH/H2O, and homogenized in a tissue-lyser (QIAGEN) at 25 Hz for 5 min. The tubes were then centrifugated at 15,000 rpm for 15 min, and 600 uL of the supernatants was collected and load on SPE cartdriges (Oasis HLB, Hydrophilic-Lipophilic-Balance, 30 mg with particle sizes of 30 um), that were prealably activated with 100% MeOH, and 100% H2O. After loading the supernatants, 95/5 MeOH/H2O was used as wash solvent (1 mL), then the elution was carried with 70/30 MeOH/H2O (2mL), an 100% methanol (1mL). Dried extracts were resuspended in 2.5 mL of 70/30 MeOH/H2O containing 0.5 uM of amitriptiline as internal standard. These extracts were used to prepare various combination of sample ratios, such as for NIST 1950 SRM / Tomato: 100/0, 75/25, 50/50, 25/75, 0/100. See the metadata for more informations. LC-MS/MS. Samples were analyzed using ultra high performance liquid chromatography (Dionex) coupled to a QTOF (Maxis II Bruker daltonics). Each sample was analyzed in triplicate, and the injection sequence was randomised. The QC mix sample was used to optimise the LC-MS/MS parameters and was injected periodically throughout the sequence. No carry over was observed. UPDATE The intensity relative standard deviation was observed at not more than 15% for replicates and QC mix samples, and the retention time deviation for the internal standards (amitriptiline m/z 278.190 and 3.57 min) was observed below 1 sec for replicates and QC mix, and not more than 2-3 sec for replicates and QC mix samples (see feature m/z 485.3657 and 660.5 sec). Liquid chromatography. Three different chromatographic conditions were used (named C18, C18RTshift and C18polar). Two chromatographic columns were used: the Phenomenex Kinetex C18 1.7 um (100A) 100 x 2.1 (for C18 and C18RTshift), and the Phenomenex Kinetex C18 polar 1.6 um (100A) 100 x 2.1 mm (for C18polar). Both columns were equipped with a C18 guard cartridge (Phenomenex).. The mobile phases consisted of A (100% H2O + 0.1% formic acid) and B (100% ACN + 0.1% fomic acid), and the flow rate was set to 0.5 uL/min throughout the experiment, and the column maintainted at 40C. The same chromatographic elution method was used for the C18 and C18 polar conditions: 0.00-0.25 min, 20% B; 0.25 - 4.00 min, 50% B; 4.00 - 15.00 min, 100% B; 15.00 -15.90 min, 100% B; 16.00 - 18.00 min, 20% B. Mass Spectrometry. UPDATE File conversion. The raw data were converted to centroid in the mzML format with MSConvert (ProteoWizard, release 201812)