ABSTRACT
Human induced pluripotent stem cells (iPSCs) can be differentiated into neurons, providing living human neurons to model brain diseases. However, it is unclear how different types of molecules work together to regulate stem cell and neuron biology in healthy and disease states. In this study, we conducted integrated proteomics, lipidomics, and metabolomics analyses with confident identification, accurate quantification, and reproducible measurements to compare the molecular profiles of human iPSCs and iPSC-derived neurons. Proteins, lipids, and metabolites related to mitosis, DNA replication, pluripotency, glycosphingolipids, and energy metabolism were highly enriched in iPSCs, whereas synaptic proteins, neurotransmitters, polyunsaturated fatty acids, cardiolipins, and axon guidance pathways were highly enriched in neurons. Mutations in the GRN gene lead to the deficiency of the progranulin (PGRN) protein, which has been associated with various neurodegenerative diseases. Using this multiomics platform, we evaluated the impact of PGRN deficiency on iPSCs and neurons at the whole-cell level. Proteomics, lipidomics, and metabolomics analyses implicated PGRN's roles in neuroinflammation, purine metabolism, and neurite outgrowth, revealing commonly altered pathways related to neuron projection, synaptic dysfunction, and brain metabolism. Multiomics data sets also pointed toward the same hypothesis that neurons seem to be more susceptible to PGRN loss compared to iPSCs, consistent with the neurological symptoms and cognitive impairment from patients carrying inherited GRN mutations.
ABSTRACT
Mass spectrometry (MS) analysis is often challenged by contaminations from detergents, salts, and polymers that compromise data quality and can damage the chromatography and MS instruments. However, researchers often discover contamination issues only after they acquire the data. There is no existing contaminant assay that is sensitive enough to detect trace amounts of contaminants from a few microliters of samples prior to MS analysis. To address this crucial need in the field, we developed a sensitive, rapid, and cost-effective contaminant spot check and removal assay (ContamSPOT) to detect and quantify trace amounts of contaminants, such as detergents, salts, and other chemicals commonly used in the MS sample preparation workflow. Only 1 µL of the sample was used prior to MS injection to quantify contaminants by ContamSPOT colorimetric or fluorometric assay on a thin layer chromatography (TLC) plate. We also optimized contaminant removal methods to salvage samples with minimal loss when ContamSPOT showed a positive result. ContamSPOT was then successfully applied to evaluate commonly used bottom-up proteomic methods regarding the effectiveness of removing detergent, peptide recovery, reproducibility, and proteome coverage. We expect ContamSPOT to be widely adopted by MS laboratories as a last-step quality checkpoint prior to MS injection. We provided a practical decision tree and a step-by-step protocol with a troubleshooting guide to facilitate the use of ContamSPOT by other researchers. ContamSPOT can also provide a unique readout of sample cleanliness for developing new MS-based sample preparation methods in the future.
