SummaryReproducibility in molecular and cellular studies is fundamental to scientific discovery. To establish the reproducibility of a well-defined long-term neuronal differentiation protocol, we repeated the cellular and molecular comparison of the same two iPSC lines across five distinct laboratories. Despite uncovering acceptable variability within individual laboratories, we detect poor cross-site reproducibility of the differential gene expression signature between these two lines. Factor analysis identifies the laboratory as the largest source of variation along with several variation-inflating confounders such as passaging effects and progenitor storage. Single-cell transcriptomics shows substantial cellular heterogeneity underlying inter-laboratory variability and being responsible for biases in differential gene expression inference. Factor analysis-based normalization of the combined dataset can remove the nuisance technical effects, enabling the execution of robust hypothesis-generating studies. Our study shows that multi-center collaborations can expose systematic biases and identify critical factors to be standardized when publishing novel protocols, contributing to increased cross-site reproducibility.

Prolonged storage of biospecimen can lead to artificially altered metabolite concentrations and thus bias data analysis in metabolomics experiments. To elucidate the potential impact of long-term storage on the metabolite profile, a pooled human plasma sample was aliquoted and stored at -80 °C. During a time period of five years, 1012 of the aliquots were measured with the Biocrates AbsoluteIDQ p180 targeted-metabolomics assay at 193 time points. Modeling the concentration courses over time revealed that 55 out of 111 metabolites remained stable. The statistically significantly changed metabolites showed on average an increase or decrease of +13.7% or -14.5%, respectively. In detail, increased concentration levels were observed for amino acids (mean: + 15.4%), the sum of hexoses (+7.9%), butyrylcarnitine (+9.4%), and some phospholipids mostly with chain lengths exceeding 40 carbon atoms (mean: +18.0%). Lipids tended to exhibit decreased concentration levels with the following mean concentration changes: acylcarnitines, -12.1%; lysophosphatidylcholines, -15.1%; diacyl-phosphatidylcholines, -17.0%; acyl-alkyl-phosphatidylcholines, -13.3%; sphingomyelins, -14.8%. We conclude that storage of plasma samples at -80 °C for up to five years can lead to altered concentration levels of amino acids, acylcarnitines, glycerophospholipids, sphingomyelins, and the sum of hexoses. These alterations must be considered when analyzing metabolomics data from long-term epidemiological studies.

IntroductionAlthough cultured cells are nowadays regularly analyzed by metabolomics technologies, some issues in study setup and data processing are still not resolved to complete satisfaction: a suitable harvesting method for adherent cells, a fast and robust method for data normalization, and the proof that metabolite levels can be normalized to cell number.ObjectivesWe intended to develop a fast method for normalization of cell culture metabolomics samples, to analyze how metabolite levels correlate with cell numbers, and to elucidate the impact of the kind of harvesting on measured metabolite profiles.MethodsWe cultured four different human cell lines and used them to develop a fluorescence-based method for DNA quantification. Further, we assessed the correlation between metabolite levels and cell numbers and focused on the impact of the harvesting method (scraping or trypsinization) on the metabolite profile.ResultsWe developed a fast, sensitive and robust fluorescence-based method for DNA quantification showing excellent linear correlation between fluorescence intensities and cell numbers for all cell lines. Furthermore, 82–97 % of the measured intracellular metabolites displayed linear correlation between metabolite concentrations and cell numbers. We observed differences in amino acids, biogenic amines, and lipid levels between trypsinized and scraped cells.ConclusionWe offer a fast, robust, and validated normalization method for cell culture metabolomics samples and demonstrate the eligibility of the normalization of metabolomics data to the cell number. We show a cell line and metabolite-specific impact of the harvesting method on metabolite concentrations.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-016-1104-8) contains supplementary material, which is available to authorized users.

Aims/hypothesisDiabetic retinopathy is a major complication of type 2 diabetes and the leading cause of blindness in adults of working age. Neuronal defects are known to occur early in disease, but the source of this dysfunction is unknown. The aim of this study was to examine differences in the retinal membrane proteome among non-diabetic mice and mouse models of diabetes either with or without metformin treatment.MethodsAlterations in the retinal membrane proteome of 10-week-old diabetic db/db mice, diabetic db/db mice orally treated with the anti-hyperglycaemic metformin, and congenic wild-type littermates were examined using label-free mass spectrometry. Pathway enrichment analysis was completed with Genomatix and Ingenuity. Alterations in Slc17a7 mRNA and vesicular glutamate transporter 1 (VGLUT1) protein expression were evaluated using real-time quantitative PCR and immunofluorescence.ResultsA total of 98 proteins were significantly differentially abundant between db/db and wild-type animals. Pathway enrichment analysis indicated decreases in levels of proteins related to synaptic transmission and cell signalling. Metformin treatment produced 63 differentially abundant proteins compared with untreated db/db mice, of which only 43 proteins were found to occur in both datasets, suggesting that treatment only partially normalises the alterations induced by diabetes. VGLUT1, which is responsible for loading glutamate into synaptic vesicles, was found to be differentially abundant in db/db mice and was not normalised by metformin. The decrease in Slc17a7/VGLUT1 was confirmed by transcriptomic and immunocytochemical analysis.Conclusions/interpretationThese findings expand the knowledge of the protein changes in diabetic retinopathy and suggest that membrane-associated signalling proteins are susceptible to changes that are partially ameliorated by treatment.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-013-3070-2) contains peer-reviewed but unedited supplementary material, which is available to authorised users.