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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
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.
| Study ID | ST002877 |
| Study Title | Metabolic Profiling of Raw264.7 Mouse Macrophage Cells Cultured with Alanine |
| Study Summary | To 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 Name | jiang |
| First Name | ming |
| Address | No. 135, Xingang Xi Road, Guangzhou, 510275, P. R. China, guangzhou, guangdong, 510006, China |
| jiangm28@mail.sysu.edu.cn | |
| Phone | 13434283781 |
| Submit Date | 2023-09-14 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | GC-MS |
| Release Date | 2023-09-26 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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 |
|---|---|---|
| SA314487 | Ala-40-2-1 | Ala |
| SA314488 | Ala-40-2-2 | Ala |
| SA314489 | Ala-40-3-2 | Ala |
| SA314490 | Ala-40-1-2 | Ala |
| SA314491 | Ala-40-3-1 | Ala |
| SA314492 | Ala-40-1-1 | Ala |
| SA314493 | control-2-1 | Control |
| SA314494 | control-1-2 | Control |
| SA314495 | control-2-2 | Control |
| SA314496 | control-3-1 | Control |
| SA314497 | control-3-2 | Control |
| SA314498 | control-1-1 | Control |
| 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 |
