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Integrating a generalized data analysis workflow with the Single-probe mass spectrometry experiment for single cell metabolomics.

ABSTRACT: We conducted single cell metabolomics studies of live cancer cells through online single cell mass spectrometry (SCMS) experiments combined with a generalized comprehensive data analysis workflow. The SCMS experiments were carried out using the Single-probe device coupled with a mass spectrometer to measure molecular profiles of cells in response to two mitotic inhibitors, taxol and vinblastine, under a series of treatment conditions. SCMS metabolomic data were analyzed using a comprehensive approach, including data pre-treatment, visualization, statistical analysis, machine learning, and pathway enrichment analysis. For comparative studies, traditional liquid chromatography-MS (LC-MS) experiments were conducted using lysates prepared from bulk cell samples. Metabolomic profiles of single cells were visualized through Partial Least Square-Discriminant Analysis (PLS-DA), and the phenotypic biomarkers associated with emerging phenotypes induced by drug treatment were discovered and compared through a series of rigorous statistical analysis. Species of interest were further identified at both the single cell and population levels. In addition, four biological pathways potentially involved in the drug treatment were determined through pathway enrichment analysis. Our work demonstrated the capability of comprehensive pipeline studies of single cell metabolomics. This method can be potentially applied to broader types of SCMS datasets for future pharmaceutical and chemotherapeutic research.

SUBMITTER: Liu R

PROVIDER: S-EPMC6579046 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Project description:BackgroundAssessing the reproducibility of measurements is an important first step for improving the reliability of downstream analyses of high-throughput metabolomics experiments. We define a metabolite to be reproducible when it demonstrates consistency across replicate experiments. Similarly, metabolites which are not consistent across replicates can be labeled as irreproducible. In this work, we introduce and evaluate the use (Ma)ximum (R)ank (R)eproducibility (MaRR) to examine reproducibility in mass spectrometry-based metabolomics experiments. We examine reproducibility across technical or biological samples in three different mass spectrometry metabolomics (MS-Metabolomics) data sets.ResultsWe apply MaRR, a nonparametric approach that detects the change from reproducible to irreproducible signals using a maximal rank statistic. The advantage of using MaRR over model-based methods that it does not make parametric assumptions on the underlying distributions or dependence structures of reproducible metabolites. Using three MS Metabolomics data sets generated in the multi-center Genetic Epidemiology of Chronic Obstructive Pulmonary Disease (COPD) study, we applied the MaRR procedure after data processing to explore reproducibility across technical or biological samples. Under realistic settings of MS-Metabolomics data, the MaRR procedure effectively controls the False Discovery Rate (FDR) when there was a gradual reduction in correlation between replicate pairs for less highly ranked signals. Simulation studies also show that the MaRR procedure tends to have high power for detecting reproducible metabolites in most situations except for smaller values of proportion of reproducible metabolites. Bias (i.e., the difference between the estimated and the true value of reproducible signal proportions) values for simulations are also close to zero. The results reported from the real data show a higher level of reproducibility for technical replicates compared to biological replicates across all the three different datasets. In summary, we demonstrate that the MaRR procedure application can be adapted to various experimental designs, and that the nonparametric approach performs consistently well.ConclusionsThis research was motivated by reproducibility, which has proven to be a major obstacle in the use of genomic findings to advance clinical practice. In this paper, we developed a data-driven approach to assess the reproducibility of MS-Metabolomics data sets. The methods described in this paper are implemented in the open-source R package marr, which is freely available from Bioconductor at http://bioconductor.org/packages/marr .

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Integrating a generalized data analysis workflow with the Single-probe mass spectrometry experiment for single cell metabolomics.

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