Summary of Study ST002875
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 PR001797. The data can be accessed directly via it's Project DOI: 10.21228/M8F422 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 | ST002875 |
| Study Title | LAT1-4F2hc Lipidomics |
| Study Summary | The LAT1-4F2hc complex (SLC7A5-SLC3A2) facilitates uptake of essential amino acids, hormones and drugs. Its dysfunction is associated with many cancers and immune/neurological disorders. Here, we apply native mass spectrometry (MS)-based approaches to provide evidence of super-dimer formation (LAT1-4F2hc)2. When combined with lipidomics, and site-directed mutagenesis, we discover four endogenous phosphatidylethanolamine (PE) molecules at the interface and C-terminus of both LAT1 subunits. We find that interfacial PE binding is regulated by 4F2hc-R183 and is critical for regulation of palmitoylation on neighbouring LAT1-C187. Combining native MS with mass photometry (MP) we reveal that super-dimerization is sensitive to pH, and modulated by complex N-glycans on the 4F2hc subunit. Free LAT1 is also present in the lysosome after interferon-γ (IFN-γ) stimulation suggesting that the assembly of LAT1-4F2hc is regulated by redox dynamics in vivo. Together our results link PTM and lipid binding with regulation and trafficking of the LAT1-4F2hc super dimer. |
| Institute | University of Oxford |
| Department | Kavli Institute for Nanoscience Discovery |
| Laboratory | Prof. Carol Robinson Group |
| Last Name | Wu |
| First Name | Di |
| Address | Kavli Institute for Nanoscience Discovery, University of Oxford, Oxford, OX1 3QU, UK |
| di.wu2@chem.ox.ac.uk | |
| Phone | +4401865275278 |
| Submit Date | 2023-09-14 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-03-14 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001797 |
| Project DOI: | doi: 10.21228/M8F422 |
| Project Title: | LAT1-4F2hc Lipidomics |
| Project Type: | MS identification |
| Project Summary: | The LAT1-4F2hc complex (SLC7A5-SLC3A2) facilitates uptake of essential amino acids, hormones and drugs. Its dysfunction is associated with many cancers and immune/neurological disorders. Here, we apply native mass spectrometry (MS)-based approaches to provide evidence of super-dimer formation (LAT1-4F2hc)2. When combined with lipidomics, and site-directed mutagenesis, we discover four endogenous phosphatidylethanolamine (PE) molecules at the interface and C-terminus of both LAT1 subunits. We find that interfacial PE binding is regulated by 4F2hc-R183 and is critical for regulation of palmitoylation on neighbouring LAT1-C187. Combining native MS with mass photometry (MP) we reveal that super-dimerization is sensitive to pH, and modulated by complex N-glycans on the 4F2hc subunit. Free LAT1 is also present in the lysosome after interferon-? (IFN-?) stimulation suggesting that the assembly of LAT1-4F2hc is regulated by redox dynamics in vivo. Together our results link PTM and lipid binding with regulation and trafficking of the LAT1-4F2hc super dimer. |
| Institute: | University of Oxford |
| Department: | Kavli Institute for Nanoscience Discovery |
| Laboratory: | Prof. Carol Robinson Group |
| Last Name: | Wu |
| First Name: | Di |
| Address: | Kavli Institute for Nanoscience Discovery, University of Oxford, Oxford, OX1 3QU, UK |
| Email: | di.wu2@chem.ox.ac.uk |
| Phone: | +4401865275278 |
Subject:
| Subject ID: | SU002988 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Cell Strain Details: | HEK293F |
| Species Group: | Mammals |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Treatment |
|---|---|---|
| SA314465 | LAT1-4F2hc_WT_delipidated_2 | Delipidated |
| SA314466 | LAT1-4F2hc_WT_delipidated_1 | Delipidated |
| SA314467 | LAT1-4F2hc_WT_2 | Untreated |
| SA314468 | LAT1-4F2hc_WT_1 | Untreated |
| Showing results 1 to 4 of 4 |
Collection:
| Collection ID: | CO002981 |
| Collection Summary: | The LAT1-4F2hc complex was buffer-exchanged into 1M ammonium acetate, pH 7.0 with 2 mM OGNG and incubated with 1 µg trypsin overnight at 37 °C. The digested peptide/lipid mixture was dried using a SpeedVac vacuum concentrator (Thermo Fisher Scientific) |
| Sample Type: | HEK cells |
Treatment:
| Treatment ID: | TR002997 |
| Treatment Summary: | The LAT1-4F2hc complex was buffer-exchanged into 1M ammonium acetate (pH 7.0) with 20 mM OGNG using a 100 kD MWCO centrifugal filter (Amicon Ultra-0.5 ml, Millipore) and incubated for 4 h at room temperature with gentle mixing. Excess lipomicelles and detergents were removed by buffer-exchanging into 1 M ammonium acetate with 2 mM OGNG using the same filter. |
Sample Preparation:
| Sampleprep ID: | SP002994 |
| Sampleprep Summary: | The lipid mixture was reconstituted with 70% mobile phase A (acetonitrile/H2O: 60/40, 10 mM ammonium formate and 0.1% formic acid) and 30% mobile phase B (isopropanol/acetonitrile: 90/10, 10 mM ammonium formate and 0.1% formic acid) for the following LC-MS/MS analysis. |
Combined analysis:
| Analysis ID | AN004712 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Dionex Ultimate 3000 |
| Column | Thermo Acclaim PepMap 100 C18 (150 x 0.007 mm, 3um, 100A) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo LTQ XL |
| Ion Mode | NEGATIVE |
| Units | Peak area |
Chromatography:
| Chromatography ID: | CH003548 |
| Chromatography Summary: | Column information: https://www.thermofisher.com/order/catalog/product/164568 |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | Thermo Acclaim PepMap 100 C18 (150 x 0.007 mm, 3um, 100A) |
| Column Temperature: | 40 |
| Flow Gradient: | 30%-99% |
| Flow Rate: | 300 nl/min |
| Solvent A: | 60% acetonitrile/40% water; 10 mM ammonium formate; 0.1% formic acid |
| Solvent B: | 90% isopropanol/10% acetonitrile; 10 mM ammonium formate; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS004458 |
| Analysis ID: | AN004712 |
| Instrument Name: | Thermo LTQ XL |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | - |
| Ion Mode: | NEGATIVE |
