Dataanalysis

Raw MS data were processed using Data Analysis (version 4.1; Bruker Daltonics). Internal mass calibration was performed with sodium formate injected in the first 40 s of each chromatographic run. Data were transferred to MZXL format and further processed with MS‐DIAL (Tsugawa et al., 2015) (v. 3.96) for annotation of the lipids, using the Lipidblast library (>35,000 MS/MS spectra) and following the shorthand nomenclature proposed by Liebisch et al. (2013) based upon the LIPID MAPS terminology (http://www.lipidmaps.org). The parameter settings were defined as follows: retention time begins at 0.5 min; retention time ends at 25 min; mass range begins at 60 Da; mass range ends at 2000 Da; MS1 (centroiding) tolerance at 0.005 Da; MS2 (centroiding) tolerance at 0.01 Da; smoothing level at 3 scans; minimum peak height at 500 amplitude; mass slice width at 0.05 Da; accurate mass tolerance (MS1) at 0.02 Da; accurate mass tolerance (MS2) at 0.02 Da; and identification score cutoff at 70%, without using retention information for scoring. Lipid concentrations were expressed in mg/kg wet weight (ww) based on lipid standards (Avanti Polar Lipids).

LC ESI MS analysis was performed as previously described^{23 (link)}. In brief, following in-gel tryptic digestion peptides were analysed using LC ESI MS analysis using a maXis UHR TOF mass spectrometer (Bruker Daltonics) using an automated acquisition approach. MS and MS/MS scans (m/z 50–3000) were acquired in positive ion mode. Lock mass calibration was performed using HP 1221.990364. Line spectral data were then processed into peak list by Data analysis (Bruker Daltonics) and the sum peak finder algorithm was used for peak detection using a signal to noise (S/N) ratio of 10, a relative to the base peak intensity of 0.1%, and an absolute intensity threshold of 100. Extracted ion chromatograms were generated for both the non-deamidated and deamidated peptide in Data analysis. MS/MS spectra of the peptides identified were manually verified.

Raw LC-MS/MS data had lock mass calibration applied and were converted into mzXML format using Bruker DataAnalysis and Bruker Compass Xport software.
The data processing software MZmine2 (version 2.37.1-corr17.7) was used for detection of chromatographic peaks and filtering of detected features (retention time—m/z pairs). Processing parameters to obtain the feature table, generating and exporting the mgf and quantification table to be used in GNPS and SIRIUS4 and for ion identity molecular networking in MZmine2 are reported in Table S2.

Commercial PMI-Byos software (Protein Metrics Inc.; v. 4.5) as well as DataAnalysis (Bruker; v. 5.0) were used for deconvoluting intact mass data. For the PMI intact mass software, a range of charge states from 5+ to 35+ and a mass range from 130 to 160 kDa were applied for deconvolution in relevant retention time windows. In DataAnalysis the deconvolution was performed using the maximum entropy algorithm with a mass range from 130 to 160 kDa, a data point spacing of 1 m/z and an instrument resolving power of 5,000. For generation of extracted ion traces (EIC) DataAnalysis was used in the case of measurements performed on the FTICR instrument, whereas Xcalibur or Freestyle (v. 1.8) was used for the generation of EICs for all data measured on Thermo MS instruments. All extracted EICs were smoothed and imported in Adobe illustrator to generate the presented figures.

The spectra acquired by nanoLC-MS/MS were converted into xml files using DataAnalysis (version 4.1; Bruker) and searched against the Swissprot (release 51.0) database using MASCOT (version 2.2.07). The MASCOT search parameters for precursor ion and fragment ion tolerance were 80 ppm and 0.07 Da, respectively. The following search parameters were selected: Taxonomy, Human; missed cleavages, 1; enzyme, trypsin; fixed modifications, carbamidomethyl (C); and variable modifications, oxidation (M) and deamidation (NQ). Peptides were considered as “identified” if their individual MASCOT ion score was higher than 25 (p < 0.01).

Five microliters of each sample were injected into an XCT6330 LC/MSD ultratrap system (Agilent Technologies) equipped with a Nucleosil 100 C18 column (3 μm × 100 mm × 2 mm internal diameter, Dr. Maisch GmbH). The column was used at 40 °C. A linear gradient was performed from 0 to 10% eluent B (0.06% formic acid in acetonitrile) over 25 min with a flow rate of 400 µl/min. The column was re-equilibrated for 10 min with 100% buffer A (0,1% formic acid in water). Ionization alternated between positive and negative ion modes with a capillary voltage of 3.5 kV at 350 °C. Extracted ion chromatograms (EICs) in negative ion mode for UDP-MurNAc-l-Ala-d-iGlu-l-Lys-d-Ala-d-Ala (m/z⁻¹ 1148.34 ± 0.1) and UDP-MurNAc-l-Ala-d-iGlu-l-Lys-(d-Asp)-d-Ala-d-Ala (m/z⁻¹ 1263.37 ± 0.1) were analyzed with Data Analysis (Bruker), exported and presented with GraphPad Prism 6.0.