Evaluation of sample preparation methods for protein analysis of liver organoids

For mass spectrometry (MS)-based proteomics, different sample preparation methods were evaluated for limited cell samples (10 000-200 000 cells), and the optimized method was used to analyze liver organoids (20-30 organoids, equivalent to approximately 50 000 cells). The sample preparation methods evaluated were urea-based in-solution-digestion (ISD-Urea), a detergent-free sample preparation method named sample preparation by easy extraction and digestion (SPEED), and a method with bead-based sample clean-up called single-pot, solid-phase enhanced sample preparation (SP3). The samples were analyzed with liquid chromatography (LC)-MS using a nanoLC column or a micropillar array column coupled to a trapped ion mobility time of flight (timsTOF)-MS. The samples prepared by SPEED had the overall highest number of protein identifications among all the sample sizes, with a range of 1684-3503 proteins identified for 10 000-100 000 cells. A quantification method for estimation of protein yield was found difficult, but because of the superior performance regarding number of protein identifications, SPEED was chosen as method for analyzing liver organoids of different metabolic states using label-free-quantification. The results showed significant differences between the organoid samples, with many of the hypothesized protein profiles found. The number of protein identifications was in the range of 2140-3203 proteins for the samples consisting of 20-30 organoids each. The method was successfully implemented as a preliminary step for disease studies, e.g. non-alcoholic fatty liver disease (NAFLD).