Summary of Study ST002877

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001799. The data can be accessed directly via it's Project DOI: 10.21228/M85M79 This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002877
Study TitleMetabolic Profiling of Raw264.7 Mouse Macrophage Cells Cultured with Alanine
Study SummaryTo identify the catabolites of L-Alanine on promoting phagocytosis, GC-MS based metabolomics analysis was adopted to explore L-Alanine-reprogrammed metabolome. The metabolic flow of the TCA cycle was dysregulated. Meanwhile, six metabolites (oleate, palmitate, stearate, myristate, arachidonate and linoleate) in biosynthesis of saturated and unsaturated fatty acids were increased upon L-Alanine treatment, where palmitate was the biggest absolute increment in abundance. Thus, L-Alanine promotes the biosynthesis of fatty acids.
Institute
Sun Yat-sen University
Last Namejiang
First Nameming
AddressNo. 135, Xingang Xi Road, Guangzhou, 510275, P. R. China, guangzhou, guangdong, 510006, China
Emailjiangm28@mail.sysu.edu.cn
Phone13434283781
Submit Date2023-09-14
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailGC-MS
Release Date2023-09-26
Release Version1
ming jiang ming jiang
https://dx.doi.org/10.21228/M85M79
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001799
Project DOI:doi: 10.21228/M85M79
Project Title:Exogenous L-Alanine promotes phagocytosis via dual regulations of TLR4 to eliminate multidrug-resistant bacterial pathogens
Project Type:MS quantitative analysis
Project Summary:Multidrug-resistant bacteria present a major threat to public health. Therefore, new drugs or approaches are urgently needed to manage and mitigate this threat. Here, we screen the molecular candidates that allow the survival of mice upon multidrug-resistant Vibrio parahaemolyticus infection by integrated proteomic and metabolomics analysis, where L-Alanine metabolism and phagocytosis are highly correlated. The role of L-Alanine on boosting mouse survival is further confirmed with in vivo bacterial challenge studies on multidrug-resistant bacteria including V. parahaemolyticus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae. Functional studies demonstrate that exogenous L-Alanine promotes phagocytosis to these different species of multidrug-resistant pathogens. The underlying mechanism involves two events that are L-Alanine-dependently increased TLR4 expression, and L-Alanine-enhanced TLR4 signaling via increasing the biosynthesis and secretion of fatty acids such as palmitate. Palmitate enhances the binding of LPS to TLR4 and thereby promotes TLR4 dimmer formation and endocytosis for the subsequent activation of PI3K/Akt and NF-κB pathways and phagocytosis of bacteria. These results suggest that modulation of metabolic environment is a plausible approach for combating infection with multidrug-resistant bacteria.
Institute:sun yat-sen university
Last Name:jiang
First Name:ming
Address:No. 135, Xingang Xi Road, Guangzhou, 510275, P. R. China, guangzhou, guangdong, 510006, China
Email:jiangm28@mail.sysu.edu.cn
Phone:13434283781

Subject:

Subject ID:SU002990
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id factor
SA314487Ala-40-2-1Ala
SA314488Ala-40-2-2Ala
SA314489Ala-40-3-2Ala
SA314490Ala-40-1-2Ala
SA314491Ala-40-3-1Ala
SA314492Ala-40-1-1Ala
SA314493control-2-1Control
SA314494control-1-2Control
SA314495control-2-2Control
SA314496control-3-1Control
SA314497control-3-2Control
SA314498control-1-1Control
Showing results 1 to 12 of 12

Collection:

Collection ID:CO002983
Collection Summary:Cells were counted, washed with cold PBS and then flash-frozen in liquid N2
Sample Type:Cultured cells

Treatment:

Treatment ID:TR002999
Treatment Summary:To trace L-Alanine metabolism, RAW264.7 cell were grown in DMEM (Hyclone) supplemented with 10% (v/v) cosmic calf (Hyclone), then transferred into L-Alanine-free medium and deprived of serum overnight. Subsequently, cells were incubated with 5 mM L-Alanine and 5mM [U-13C]-L-Alanine in serum-starved medium (DMEM/0.5% serum). Additionally, fresh media containing L-Alanine and [U-13C]-L-Alanine were exchanged 2 h before metabolite extraction for metabolic analysis.

Sample Preparation:

Sampleprep ID:SP002996
Sampleprep Summary:Cells were homogenized with the first solvent (the mixture of chloroform, methanol and water (1:2:1, v/v/v)) for 30 s at 4 °C and then centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was collected and deposit was re-homogenized with the second solvent (methanol alone) before a second centrifugation. The 2 supernatants were mixed, and aliquot of sample was transferred to a GC sampling vial containing 5 μL 0.1 mg/mL ribitol (Sigma) as an analytical internal standard and then dried in a vacuum centrifuge concentrator before the subsequent derivatization. A total of 2 technical replicates were prepared for each sample.

Combined analysis:

Analysis ID AN004714
Analysis type MS
Chromatography type GC
Chromatography system Thermo Scientific Trace GC Ultra with DSQ II GC/MS
Column Agilent DB5-MS (30m x 0.25mm, 0.25um)
MS Type EI
MS instrument type Triple quadrupole
MS instrument name Thermo Scientific Trace GC Ultra with DSQ II GC/MS
Ion Mode POSITIVE
Units Peak area

Chromatography:

Chromatography ID:CH003550
Instrument Name:Thermo Scientific Trace GC Ultra with DSQ II GC/MS
Column Name:Agilent DB5-MS (30m x 0.25mm, 0.25um)
Column Temperature:270 °C
Flow Gradient:none
Flow Rate:1.0 mL/min
Solvent A:none
Solvent B:none
Chromatography Type:GC

MS:

MS ID:MS004460
Analysis ID:AN004714
Instrument Name:Thermo Scientific Trace GC Ultra with DSQ II GC/MS
Instrument Type:Triple quadrupole
MS Type:EI
MS Comments:samples was derivatized and then used to firstly protect carbonyl moieties through methoximation, through a 90 min 37 ℃ reaction with 40 μL of 20 mg/mL methoxyamine hydrochloride (Sigma-Aldrich) in pyridine, followed by derivatization of acidic protons through a 30 min 37 0C reaction with the addition of 80 μL N-methyl-N-trimethylsilyltrifluoroace-tamide (MSTFA, Sigma-Aldrich). The derivatized sample of 1 μL was injected into a 30m × 250 μm i.d. × 0.25 μm DBS-MS column using splitless injection and analysis was carried out by Trace DSQ II (Thermo Scientific). The initial temperature of the GC oven was held at 85 0C for 5 min followed by an increase to 330 0C at a rate of 15 0C min-1 then held for 5 min. Helium was used as carrier gas and flow was kept constant at 1 mL min-1. The MS was operated in a range of 50-600 m/z.
Ion Mode:POSITIVE
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