Summary of Study ST004049
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 PR002539. The data can be accessed directly via it's Project DOI: 10.21228/M8FR75 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 | ST004049 |
| Study Title | Comparison of lipidome from phagosomes containing Pam3csk4-beads vs. uncoupled-beads |
| Study Type | Lipidomics |
| Study Summary | Goals of the study: Lipids can participate in signaling cascades. LC3-associated phagocytosis (LAP) facilitates cargo elimination during phagocytosis. Lipid signaling during LC3-associated phagocytosis remains understudied. The goal of the study is to evaluate differences in lipid composition at the phagosomal membrane when phagocytes are fed inert beads (biotin labelled) compared with beads labelled with the TLR2 agonist Pam3csk4 that induces LAP. Method: We analyzed isolated phagosomes (~20x10^6/sample) from immortalized bone marrow derived macrophages (iBMDM) fed Pam3csk4-conjugated beads (6 samples) or uncoupled-beads (3 samples). Phagosomes were isolated 1h after phagocytosis stimulation using mechanical disruption of cells and sucrose gradients. Results: The lipid composition differed between the groups with an overall increased in phosphatidylserine species observed in phagosomes containing Pam3csk4-beads. Phosphatidylserine (PS) species were enriched in LAP-induced phagosomes. PS species serve as signaling molecules attracting and docking Rubicon, a key component in the LAP cascade. Limiting PS enrichment at phagosomes using chemical, genetic methods or impeding Rubicon binding to PS blocks LAP progression. Even though LC3-associated phagocytosis has been implicated in disease models such as infectious diseases and cancer, this work is a basic science study not involved directly with diseases or treatments. |
| Institute | St Jude Children's Research Hospital |
| Department | Immunology |
| Laboratory | Green Lab |
| Last Name | Palacios |
| First Name | Gustavo |
| Address | 262 Danny Thomas Place, Memphis, Tennessee, 38105, USA |
| Gustavo.Palacios@stjude.org | |
| Phone | 901-595-4448 |
| Submit Date | 2025-06-04 |
| Num Groups | 2 |
| Total Subjects | 9 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2025-08-11 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002539 |
| Project DOI: | doi: 10.21228/M8FR75 |
| Project Title: | Comparison of lipidome from phagosomes containing Pam3csk4-beads vs. uncoupled-beads |
| Project Type: | Lipidomics |
| Project Summary: | Goals of the study: Lipids can participate in signaling cascades. LC3-associated phagocytosis (LAP) facilitates cargo elimination during phagocytosis. Lipid signaling during LC3-associated phagocytosis remains understudied. The goal of the study is to evaluate differences in lipid composition at the phagosomal membrane when phagocytes are fed inert beads (biotin labelled) compared with beads labelled with the TLR2 agonist Pam3csk4 that induces LAP. Method: We analyzed isolated phagosomes (~20x10^6/sample) from immortalized bone marrow derived macrophages (iBMDM) fed Pam3csk4-conjugated beads (6 samples) or uncoupled-beads (3 samples). Phagosomes were isolated 1h after phagocytosis stimulation using mechanical disruption of cells and sucrose gradients. Results: The lipid composition differed between the groups with an overall increased in phosphatidylserine species observed in phagosomes containing Pam3csk4-beads. Phosphatidylserine (PS) species were enriched in LAP-induced phagosomes. PS species serve as signaling molecules attracting and docking Rubicon, a key component in the LAP cascade. Limiting PS enrichment at phagosomes using chemical, genetic methods or impeding Rubicon binding to PS blocks LAP progression. Even though LC3-associated phagocytosis has been implicated in disease models such as infectious diseases and cancer, this work is a basic science study not involved directly with diseases or treatments. |
| Institute: | St. Jude Children's Research Hospital |
| Department: | Immunology |
| Laboratory: | Green Lab |
| Last Name: | Palacios |
| First Name: | Gustavo |
| Address: | 262 Danny Thomas Place, Memphis, Tennessee, 38105, USA |
| Email: | Gustavo.Palacios@stjude.org |
| Phone: | 901-595-4448 |
| Funding Source: | NIH |
| Contributors: | Emilio Boada-Romero, Clifford S. Guy, Gustavo Palacios, Luigi Mari, Suresh Poudel, Zhenrui Li, Piyush Sharma, Douglas R. Green* |
Subject:
| Subject ID: | SU004195 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Gender: | Not applicable |
Factors:
Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Treatment |
|---|---|---|---|
| SA467707 | EB-00_Pos | Blank | Blank |
| SA467708 | EB-00_Neg | Blank | Blank |
| SA467709 | EB-01_Pos | iBMDM | Mock |
| SA467710 | EB-02_Pos | iBMDM | Mock |
| SA467711 | EB-03_Neg | iBMDM | Mock |
| SA467712 | EB-03_Pos | iBMDM | Mock |
| SA467713 | EB-01_Neg | iBMDM | Mock |
| SA467714 | EB-02_Neg | iBMDM | Mock |
| SA467715 | EB-07_Pos | iBMDM | Pam3-Beads |
| SA467716 | EB-09_Pos | iBMDM | Pam3-Beads |
| SA467717 | EB-09_Neg | iBMDM | Pam3-Beads |
| SA467718 | EB-08_Pos | iBMDM | Pam3-Beads |
| SA467719 | EB-08_Neg | iBMDM | Pam3-Beads |
| SA467720 | EB-06_Neg | iBMDM | Pam3-Beads |
| SA467721 | EB-07_Neg | iBMDM | Pam3-Beads |
| SA467722 | EB-06_Pos | iBMDM | Pam3-Beads |
| SA467723 | EB-05_Pos | iBMDM | Pam3-Beads |
| SA467724 | EB-04_Pos | iBMDM | Pam3-Beads |
| SA467725 | EB-04_Neg | iBMDM | Pam3-Beads |
| SA467726 | EB-05_Neg | iBMDM | Pam3-Beads |
| Showing results 1 to 20 of 20 |
Collection:
| Collection ID: | CO004188 |
| Collection Summary: | Phagocytes (~25x10^6 cells/plate, two 15cm plates per experimental point) were fed with Pam3csk4-beads or control beads so that each cell engulfed 0-3 phagosomes after 45 minutes. Plates were thoroughly washed twice with ice-cold 1X PBS to eliminate non-phagocyted beads. Cells were harvested in ice-cold 1X PBS and mechanically homogenized in 2ml 8% (w/v) sucrose (0.25M; Sigma, S9378) using glass douncers or 1 ml 27G syringes (BD, 309623). After homogenization, the disrupted cell solution was mixed with a saturated 62% (w/v) sucrose solution (1.81 M) to achieve a final sucrose concentration of ~40%. This mixture was then layered onto a 62% sucrose solution in polycarbonate centrifuge tubes (Beckman Coulter, 34058). Sequential layers of 35% (1.02 M), 25% (0.73 M), and 10% (0.29 M) sucrose solutions were carefully added on top of the cell solution mixture. All sucrose-containing buffers were prepared in 3 mM imidazole [pH 7.4] (Sigma, I202), and each layer was 8 ml, except for the 62% sucrose layer at the bottom, which was 5 ml. Equilibrated tubes were ultra-centrifuged at 100,000xg for 1h at 4ºC using a swinging-bucket rotor (SW 32Ti, Beckman Coulter, 369650; Optima XE, Beckman Coulter). Phagosome-containing beads float at the interface between the 10% and 25% sucrose layers. The bead-containing phagosomes were carefully collected, washed with cold 1X PBS (or 0.9% NaCl solution for lipidomics), and ultracentrifuged again at 100,000 × g for 30 minutes at 4°C. For lipidomic assays, bead-containing phagosomes were resuspended in 0.9% NaCl (prepared in HPLC-grade water), counted, and the same number of phagosomes were pelleted at 100,000xg for 20 min at 4ºC using fixed angle rotor (TLA-100 rotor, Beckman Coulter, 349481) in a benchtop ultracentrifuge (OptimaTL, Beckman Coulter). The pelleted phagosomes were transferred to lipidomics-compatible tubes in a minimal residual volume and flash-frozen in liquid nitrogen for further processing. |
| Sample Type: | Macrophages |
Treatment:
| Treatment ID: | TR004204 |
| Treatment Summary: | Immortalized bone marrow derived macrophages were fed Pam3csk4-conjugated beads (6 samples) or uncoupled-beads (3 samples) |
Sample Preparation:
| Sampleprep ID: | SP004201 |
| Sampleprep Summary: | Equal numbers of phagosomes (~20x106) were washed with ice-cold NaCl 0.9% saline solution, flash-frozen in liquid nitrogen, and stored at –80ºC until lipid extraction. Total lipids were extracted using a modified Folch extraction procedure (Folch, J., Lees, M. & Sloane Stanley, G. H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226, 497-509 (1957)). Briefly, 1 ml of chloroform-methanol (2:1, v/v) was added to the samples and mixed by vortexing. Then, 200 µl of 0.9% NaCl was added, and the mixture were homogenized for 30 sec at 8 m/s in a Bead Ruptor Elite (OMNI International). After incubation at room temperature for 30 sec, the samples centrifuged for 10 min at 21,000xg at 4ºC. The lower organic-phase layer was carefully transferred to a new tube and evaporated to dryness under a stream of liquid nitrogen. The dried lipid extracts were thoroughly re-dissolved with 30 µl of chloroform-methanol (2:1, v/v), transferred to autosampler vials, and analyzed by LC-MS/MS (10 µl per injection). |
| Sampleprep Protocol Filename: | Protocol_of_Untargeted_Lipidomics_09212020.pdf |
Chromatography:
| Chromatography ID: | CH005086 |
| Chromatography Summary: | Untargeted Lipidomics. Separated injections for acquiring Positive and Negative ion modes, generating two MS datafiles per sample |
| Methods Filename: | Protocol_of_Untargeted_Lipidomics_09212020.pdf |
| Instrument Name: | Vanquish Horizon |
| Column Name: | Thermo Accucore C30 (250 x 2.1mm, 2.6um) |
| Column Temperature: | 50 |
| Flow Gradient: | Time(min), %B: 0.0, 45; 4.5, 60; 5.0, 70; 8.0, 70; 19.0, 75; 20.0, 90; 33.0, 95; 34.0, 100; 39.0 100; 40.0, 45 |
| Flow Rate: | 250 µL/min |
| Solvent A: | 60% Water/40% Acetonitrile; 10mM ammonium acetate |
| Solvent B: | 10% Acetonitrile/90% Isopropyl alcohol; 10mM ammonium acetate |
| Chromatography Type: | Reversed phase |
Analysis:
| Analysis ID: | AN006694 |
| Laboratory Name: | Immuno-Metabolomics Core |
| Analysis Type: | MS |
| Acquisition Date: | 21-Sep-2020 |
| Acquisition Parameters File: | Protocol_of_Untargeted_Lipidomics_09212020.pdf |
| Software Version: | Thermo Xcalibur 4.0 |
| Operator Name: | Gustavo Palacios |
| Detector Type: | ESI-MS |
| Data Format: | .RAW |
| Chromatography ID: | CH005086 |
| Num Factors: | 3 |
| Num Metabolites: | 1216 |
| Units: | Peak area |
| Analysis ID: | AN006695 |
| Laboratory Name: | Immuno-Metabolomics Core |
| Analysis Type: | MS |
| Acquisition Date: | 21-Sep-2020 |
| Acquisition Parameters File: | Protocol_of_Untargeted_Lipidomics_09212020.pdf |
| Software Version: | Thermo Xcalibur 4.0 |
| Operator Name: | Gustavo Palacios |
| Detector Type: | ESI-MS |
| Data Format: | .RAW |
| Chromatography ID: | CH005086 |
| Num Factors: | 3 |
| Num Metabolites: | 640 |
| Units: | Peak area |
