Transomics software

The process described in Yang et al. and Lin et al. [6 (link),46 (link)] consisted of three steps. (1) The third passage of iPSC-derived RPE cell lines were treated with and without A2E. Three biological replicates were prepared representing three separate cultures derived from each cell line and were also performed separately for A2E-treated samples. (2) Proteins were extracted from each cell line, reduced and alkylated before tryptic digestion, and RapiGest was cleaved with acid. The resulting peptides were analysed using a Synapt G2 quadrupole-time-of-flight mass spectrometer (Waters Corp.) with MSE data-independent scanning. (3) Initial data were processed using ProteinLynx Global Server (Version 2.5 RC9, Waters Corp.). Further analysis was performed with TransOmics software (Waters Corp.) and the NCBI database of human sequences.

All the LC-MS raw files were converted to TransOmics software (Waters, Millford, MA, USA). Differences in the amounts of molecules between groups were analysed using SPSS Statistics 19.0 (SPSS, Chicago, IL, USA). The p values less than 0.05 were considered significant.

All solvents were LC/MS grade and purchased from Biosolve. Per experiment, ten pooled E8.5 embryos (Pgp^WT/WT or Pgp^D34N/D34N; ~50 μg total protein) were extracted with 600 μL 75% methanol. Samples were homogenised in a ball mill equipped with zirconium oxide grinding balls (MM 301 Mixer Mill; Retsch GmbH). Lipids were extracted using tert-butylmethylether (TBME) as described53 (link) and analysed by LC/MS as published previously54 (link). Processing of chromatograms, peak detection and integration were performed using MassLynx software (version 4.1, Waters). TransOmics software (Waters) was used for data preprocessing for untargeted metabolomics and for marker identification. Glycerolipid profiling was conducted using the in-house developed software RLA-Tool (A.F. and M.J.M., unpublished). Defined lipid species were searched for based on their exact m/z in the low energy function and two integral fragments (fatty acyl side chains) in the high energy function (error tolerance, 3 mDa). The identification was confirmed by the linear coherence between retention time and m/z. The identified species were then assembled into an automated method within QuantLynx embedded in MassLynx for the systematic integration of the identified lipid species in the extracts.