DOI: 10.5281/zenodo.1480658
title: CliqueMS Validation Data

description: CliqueMSData Data used for the validation of CliqueMS (https://CRAN.R-project.org/package=cliqueMS) This repository contains the mzXML files corresponding to the novel LC-MS1 spectra used to validate CliqueMS. The raw data corresponding to these files has been obtained as explained below. The corresponding mzXML files have been obtained using the msconvert tool in ProteoWizard. This repository includes 3 files: standards.mzXML positive.mzXML negative.mzXML <strong>standards.mzXML - Mixture of standards</strong> <em>Materials.</em> MS1 grade acetonitrile (ACN), ammonium acetate (NH4Ac) and NH4OH were purchased from SDS (Peypin, France). Water was produced in an in-house Milli-Q purification system (Millipore, Molsheim, France). Formic acid and ammonium fluoride were purchased from Sigma-Aldrich (Steinheim, Germany). Standards: (-)riboflavine, 1,2-distearoyl-sn-glycero-3-phosphocholine, biotin, cholic acid, deoxycholic acid, L-methionine sulfoxide, thymine and uracil were purchased from Sigma-Aldrich (Steinheim, Germany). Standard mix preparation. All standards were pooled to a final concentration of 1ppm in H2O:ACN (5:95) with 0.1% formic acid. <em>MS1 analyses.</em> MS1 analyses were performed using an UHPLC system (1290 series, Agilent Technologies) coupled to a 6550 ESI-QTOF MS (Agilent Technologies) operated in positive (ESI+) electrospray ionization mode. A vial containing the standard mix was kept at -20ºC prior to MS1 analysis. Metabolites were separated using an Acquity UPLC BEH HILIC column (2.1 x 150 mm, 1.8 \mu m) and the solvent system was A1 = 20mM ammonium acetate and 15 mM NH4OH in water and B1 = 95% ACN and 5% H2O. The linear gradient elution started at 100% B (time 0--2 min) and finished at 75% A (10-15 min). The injection volume was 5 \mu L. ESI conditions: gas temperature, 150ºC; drying gas, 13 L min--1; nebulizer, 35 psig; fragmentor, 400 V; and skimmer, 65 V. The instrument was set to acquire over the m/z range 100--1500 in full-scan mode with an acquisition rate of 4 spectra/sec. MS/MS was performed in targeted mode, and the instrument was set to acquire over the m/z range 50--1000, with a default isolation width (the width half-maximum of the quadrupole mass bandpass used during MS/MS precursor isolation) of 4 m/z. The collision energy was fixed at 20 V. <strong>positive.mzXML & negative.mzXML - Complex Samples</strong> <em>Materials.</em> MS1 grade methanol (MeOH) and acetonitrile (ACN) and analytical grade chloroform (CHCl3) were purchased from SDS (Peypin, France). Water was produced in an in-house Milli-Q purification system (Millipore, Molsheim, France). Formic acid and ammonium fluoride were purchased from Sigma-Aldrich (Steinheim, Germany). <em>Experimental Animals.</em> Irs-2-deficient mice were generated initially on a C57BL6/J: SV129 background and then backcrossed to establish a pure C57BL6/J background. Thus, the offspring resulting from the breeding of Irs-2(2/2) with RIP-Irs-2 line were C57BL6/J. <em>Metabolite extraction method.</em> Retinas were first lyophilized and metabolites were extracted adding 190 uL of MeOH and 120 uL of H2O, then vortex during 30 seconds. Afterwards, samples were frozen during 1 min in liquid nitrogen (N2) and thawed by cold sonication during 30 seconds. This step was applied three times. Then 380 uL of chloroform were added and vortexed during 30 seconds. Finally, samples were centrifuged (15000 rpm, 15 min a 4ºC). The supernatant was extracted and dried. The sample was suspended in 100 uL of H2O:MeOH (1:1) and stored at -80ºC until further analysis. MS1 analyses. MS1 analyses were performed using an UHPLC system (1290 series, Agilent Technologies) coupled to a 6550 ESI-QTOF MS (Agilent Technologies) operated in positive (ESI+) or negative (ESI-) electrospray ionization mode. Vials containing extracted metabolites were kept at -20ºC prior to MS1 analysis. When the instrument was operated in positive ionization mode, metabolites were separated using an Acquity UPLC (HSS T3) C18 reverse phase (RP) column (2.1 x 150 mm, 1.8 \mu m) and the solvent system was A1 = 0.1% formic acid in water and B1 = 0.1% formic acid in acetonitrile. When the instrument was operated in negative ionization mode, metabolites were separated using an Acquity UPLC (BEH) C18 RP column (2.1 x 150 mm, 1.7 \mu m) and the solvent system was A2 = 1 mM ammonium fluoride in water and B2 = acetonitrile. The linear gradient elution started at 100% A (time 0--2 min) and finished at 100% B (10-15 min). The injection volume was 5 \mu L. ESI conditions: gas temperature, 150ºC; drying gas, 13 L min--1; nebulizer, 35 psig; fragmentor, 400 V; and skimmer, 65 V. The instrument was set to acquire over the m/z range 100--1500 in full-scan mode with an acquisition rate of 4 spectra/sec. MS/MS was performed in targeted mode, and the instrument was set to acquire over the m/z range 50--1000, with a default isolation width (the width half-maximum of the quadrupole mass bandpass used during MS/MS precursor isolation) of 4 m/z. The collision energy was fixed at 20 V. positive.mzXML and negative.mzXML files correspond to LC-MS1 spectra obtained for positive and negative ionization, respectively.