Freshly isolated PHH were compared to liver tissue, 3D PHH spheroids after aggregation (7 d) and cells cultured as 2D monolayers (24 h and 7 d) from the same donors. Cells were washed, scraped and pelleted in phosphate buffer (pH 7.4). Subsequently, cells were resuspended in a volume of 0.5 M TEAB/0.1% SDS equivalent to cell pellet volume. Liver samples (50–100 mg) were homogenised in 0.5 M TEAB/0.1% SDS using a Mixer Mill 220 (Retsch, Haan, Germany). Cell and liver samples were subjected to one freeze-thaw cycle, sonicated and centrifuged. 100 μg protein/sample were denatured, reduced and treated with methyl methanethiosulfonate according to the manufacturer’s protocol (Sciex, Framingham, MA, USA), before being labelled with isobaric tags for absolute and relative quantification (iTRAQ), pre-fractionated by cation exchange chromatography and analysed on a Triple TOF 5600 (Sciex) as previously described43 (link). Samples from each donor were analysed in a single iTRAQ run. Data were searched using ProteinPilot 4.2 and the Paragon algorithm (Sciex) against the SwissProt database and a reversed decoy database and only proteins lying within a 1% global false discovery rate (FDR) were taken forward for analysis.
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