DOI: 10.7280/d1r70j
title: Pathway enrichments from untargeted metabolomics of plasma of Peromyscus leucopus and Mus musculus with or without LPS treatment

description: Animals that are competent reservoirs of zoonotic pathogens commonly
 suffer little morbidity from the infections. To investigate mechanisms of
 this tolerance of infection, we used single-dose lipopolysaccharide (LPS)
 as an experimental model of inflammation and compared the responses by two
 rodents: Peromyscus leucopus, white-footed deermouse and reservoir for
 agents of Lyme disease and other zoonoses, and the house mouse Mus
 musculus. Four hours after injection with LPS or saline, blood, spleen and
 liver samples were collected and subjected to RNA-seq, metabolomics, and
 specific RT-qPCR. Untargeted metabolomics identified several pathways that
 deermice and mice have in common in their responses to LPS, but also
 pathways in which they differed. The accompanying dataset is of 72
 pathways which were identified in the plasma of both P. leucopus and M.
 musculus and those that were enriched in one species or another in
 response to LPS and those that were not differentially enriched.
 The Kyoto Encyclopedia of Genes and Genomes (KEGG;
 https://www.genome.jp/kegg/) terms for the compounds of a particular
 pathway and that were identified in P. leucopus and/or in M. musculus are
 listed.
description: Adult outbred P. leucopus of the LL stock were obtained from the
 Peromyscus Genetic Stock Center (PGSC) of the University of South
 Carolina. The LL stock colony was founded with 38 animals captured near
 Linville, NC between 1982 and 1985 and has been closed since 1985.
 Sib-matings are avoided, and complete pedigree records are kept. Animals
 were maintained in the AAALAC-accredited U.C. Irvine vivarium with 2-5
 animals per cage according to sex and on 12 hours light-12 hours dark
 lighting schedule, temperature of 21-23° C, humidity of 30-70%, water ad
 libitum, and a diet of 8604 Teklad Rodent (Harlan
 Laboratories).  Animals were anesthetized with
 isoflurane and injected i.p. with a single dose of E. coli O111:B4 LPS at
 a concentration of 10 mg/kg body weight in a 50 µl volume. The control
 group was anesthetized and then injected with the 0.9% saline alone. The
 experiment started at 0800 h with 10 min intervals between animals and
 with alteration of LPS and control injections. At 4.0 hr after their
 injection the animals were euthanized. After opening the chest,
 exsanguination was performed by cardiac puncture and blood was transferred
 to a heparin sulfate coated tube (Becton-Dickinson Microtainer).
 Anticoagulated blood was centrifuged to pellet blood cells for 3 min at
 4600 x g at 4° C. Plasma and blood pellet was kept separately at -80° C
 until further analysis.  Untargeted detection and
 analysis of metabolites in plasma of P. leucopus and M. musculus were
 carried out. In brief, 40 µl volumes of plasma, which had been stored
 frozen at -80° C, were extracted with 120 µl methanol containing as
 internal standards phenylalanine d5 (175 ng/mL), 1-methyl tryptophan (37.5
 ng/mL), and arachidonoyl amide (30 ng/mL). After precipitated proteins
 were removed by centrifugation, the supernatant was dried under vacuum and
 then suspended in 50% methanol. Aliquots of 10 µl were subjected to high
 pressure liquid chromatography (HPLC) and quadrupole (Q) time-of-flight
 (TOF) mass spectrometry (MS) with periodic inclusion of pooled samples for
 quality control. Metabolites were separated on an Agilent Technologies
 Poroshell C8 column (100 x 2.1 mm, 2.7 µm) with a gradient of acetonitrile
 and water both containing 0.1% formic acid with an Agilent 1260 HPLC pump.
 The eluent was introduced into an Agilent Technologies 6520 Q-TOF-MS
 instrument equipped with an electrospray ionization source. The parameters
 for the analysis were a capillary voltage of 4000 V, fragmenter voltage of
 120 V, gas at 310° C, gas flow of 10 L/min, and nebulizer pressure of 45
 psig. Data were acquired in the positive ion mode at a scan range of
 75-1700 for the mass-to-charge ratio (m/z) and at a rate of 1.67 spectra
 per second. The raw data were deposited at the Metabolomics Workbench
 repository (https://www.metabolomicsworkbench.org) under Study ID
 ST001654.  The raw data files were converted to the
 XML-based mzML format with Proteowizard. The files were then processed for
 peak picking, grouping, and retention time correction with the XCMS suite
 of software. The centWave algorithm was applied to detect chromatographic
 peaks with these parameter settings: ≤ 30 ppm for m/z deviation in
 consecutive peaks, signal to noise ratio of 10, a prefilter of 3 scans
 with peak intensity of ≥ 750, and 10 to 45 s for peak width. Molecular
 features (MF), defined by m/z and retention time, were grouped across
 samples using a bandwidth of 15 and overlapping m/z slice of 0.02.
 Retention time correction was performed with the retcor algorithm of XCMS.
 Finally, peak area was normalized by median fold normalization
 method.  Pathway enrichment analysis was performed with
 Mummichog network analysis software v. 2, which predicts functional
 activity from spectral feature tables without a priori identification of
 metabolites. This was implemented on-line at Metaboanalyst
 (http://www.metaboanalyst.ca). The parameter settings were mass accuracy
 of 20 ppm, positive ion mode, and cut-off p value of 0.05 for the
 Fisher's exact test of observed and expected hits of a given pathway.
 Pathway enrichment in Kyoto Encyclopedia of Genes and Genomes (KEGG;
 https://www.genome.jp/kegg/) terms was determined separately for the P.
 leucopus and M. musculus sets of LPS-treated and control
 animals.  The raw data were deposited at the
 Metabolomics Workbench repository (https://www.metabolomicsworkbench.org)
 under Study ID ST001654. Molecular features data for the 40 individual
 plasma samples was deposited with the Dryad data repository
 (https://doi.org/10.7280/D12M4N). 
description: The dataset is a single Excel spreadsheet entitled "Table.
 Pathway enrichments from untargeted metabolomics of plasma of Peromyscus
 leucopus and Mus musculus with or without LPS treatment." The order
 of the column variables (with abbreviations) in the table from left to
 right are as follows: pathway, pathway total number of compounds, number
 of Mus musculus (Mus) hits, Mus significant hits, Mus expected hits, Mus
 enriched number (the number of observed hits above expected), Mus Fisher
 exact test (FET) p value, Peromyscus leucopus hits, Peromyscus significant
 hits, Peromyscus expected hits, Peromyscus enriched number, Peromyscus FET
 p value, list of KEGG terms for Mus musculus hits, list of KEGG terms
 for Peromyscus hits.