Masshunter quantitative analysis software

All MRM data were processed using Agilent MassHunter Quantitative Analysis software (Agilent B.07.00) with the Agilent Integrator algorithm for peak integration set with default values. All integrated peaks were manually inspected to ensure correct peak detection and accurate integration. For data interpretation of nucleotide sugars, normalization was performed of peak area over the total peak area of all nucleotide sugars to obtain relative abundances. Differential analyses were performed between conditions and visualized using GraphPad Prism 5.03 or Microsoft Office Excel 2007.
For dynamic tracing of isotopically labeled N-acetylglucosamine, the abundance of labeled fraction of each investigated metabolite was calculated using the ratio of ¹³C/¹²C isotopes of a specific metabolite.

To 100 μL of plasma 300 μL of cold methanol and 5 μL of internal standard (Deuterium labeled compounds) were added. After centrifugation at 900 g for 15 min at 4°C, supernatants were transferred into 2 mL 96-well deep plates and diluted in H₂O to 2 mL. Samples were then submitted to solid phase extraction (SPE) using OASIS HLB 96-well plate (30 mg/well, Waters) pretreated with MeOH (1mL) and equilibrated with 10% MeOH (1 mL). After sample application, extraction plate was washed with 10% MeOH (1 mL). After drying under aspiration, lipids mediators were eluted with 1 mL of MeOH. Prior to LC-MS/MS analysis, samples were evaporated under nitrogen gas and reconstituted in 10 μL on MeOH.
LC-MS/MS analyses of eicosanoids were performed as described [20 (link)]. Briefly, lipid mediators were separated on a ZorBAX SB-C18 column (2.1 mm, 50 mm, 1.8 μm) (Agilent Technologies) using Agilent 1290 Infinity HPLC system (Technologies) coupled to an ESI-triple quadruple G6460 mass spectrometer (Agilent Technologies). Data were acquired in Multiple Reaction Monitoring (MRM) mode with optimized conditions (ion optics and collision energy). Peak detection, integration and quantitative analysis were done using Mass Hunter Quantitative analysis software (Agilent Technologies) based on calibration lines built with commercially available eicosanoids standards (Cayman Chemicals).

Raw MRM data were analyzed using the Agile2 integrator algorithm in the MassHunter Quantitative Analysis software (version B.07.00; Agilent Technologies, CA) using the Agilent integrator algorithm for peak integration. For each peptide transition, the accuracy of the peak selection and integration were determined based on retention time, peak width, and signal response. The “best” interference-free transition of one “natural” (NAT) peptide, that is, the one with the most intense MRM signal, was used for protein quantitation. Interference assessment was performed in plasma through signal-intensity ratio measurements on the NAT and SIS peptide transitions. Protein concentrations in ng/mL were calculated by taking into account the protein's molecular weight (in g/mol; obtained from ExPASy's “pI/Mw tool” [http://web.expasy.org/compute_pi/]), the peptide's relative response (NAT/SIS), the corrected SIS peptide concentration (i.e., corrected with the composition and the purity values, as determined by amino acid analysis and capillary zone electrophoresis), and a conversion factor (of 1000).^{[35 (link)]} In cases where multiple peptides were quantified for a given protein, the one that provided the highest protein concentration was used as the quantifier.

Pancreatic islets were prepared (SI Appendix) and analyzed using an Agilent 1290 series Ultra-high-performance liquid chromatography system connected to an Agilent 6495 QQQ mass spectrometer after separation on a ZORBAX Eclipse plus C18 column (2.1 × 50 mm, 1.8 µm, 95 Å, Agilent) at 40 °C (SI Appendix). Mass spectrometry analysis was performed in positive ion mode with dynamic, scheduled multiple reaction monitoring (MRM). Mass spectrometry settings, LC-MS gradient, and MRM transitions for each lipid class were adapted from a previously published method (63 (link)). Data analysis was performed on Agilent MassHunter Quantitative analysis software. Relative quantitation was based on one-point calibration with Ceramide d18:1/8:0. The data were further normalized to the total number of islets in each sample.

After the chromatogram inspection, the obtained data were treated with the MassHunter Quantitative Analysis software (Agilent MassHunter Quantitative Analysis version 10.0) for the determination of the area of each peak. Microsoft Office Excel was used for quantitation and blank subtraction. The p-values (the t-test or the Wilcoxon/Mann–Whitney U test, Microsoft Office Excel, and SPSS, respectively) were calculated for each metabolite. Log₂FC was calculated according to the following formula:
Multivariate statistics were also performed in this study. Supervised orthogonal partial least square-discriminant analysis (OPLS-DA) was performed. Volcano plots plotting variable importance in projection (VIP) in the OPLS-DA model against corrected p-values [p(corr), loading values scaled as correlation coefficients values] were generated. Variables with absolute p(corr) lower than 0.3 show a low correlation, while variables between 0.3 and 0.5 show an intermediate correlation. Metabolites with p-values < 0.05, VIP score > 1, and p(corr) ≥ 0.3 were considered significant.
The receiver operating characteristic (ROC) curves and the area under the curve (AUC) for the studied metabolites were obtained in Metaboanalyst version 5.0 (30 (link)).

Derivatized samples were analyzed by GC-MS (Agilent 5977 Series GC/MSD, Agilent Technologies, Santa Clara, CA, USA) [15 (link)]. Trimethylsilyl derivatives (1 μL) were injected into an injector at 230 °C in splitless mode. The oven temperature ramp applied was 80 °C (initial temperature), held for 2 min, heated at 15 °C.min⁻¹ to 330 °C, and held for 6 min. The electron impact ionization mass spectrometer was set at ionization voltage 70 eV; ion source temperature 250 °C; injection port temperature 250 °C; and mass scan range 70–600 m/z at 20 scans.s⁻¹. The column used was an HP5ms column (30 m × 0.25 m × 0.25 μm). The flow rate of helium gas was 2 mL.min⁻¹. Acquisition, deconvolution, and analyses of experimental data were processed by MassHunter Quantitative Analysis software (Agilent, CA, EUA). The NIST mass spectral library (NIST 2011, Gaithersburg, MD, USA) was used for retention index (RI) comparison and data validation. Some of the identified metabolites were also confirmed by mass spectral comparison with the authentic external standards previously described.