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.

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Study IDST002875
Study TitleLAT1-4F2hc Lipidomics
Study SummaryThe 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
DepartmentKavli Institute for Nanoscience Discovery
LaboratoryProf. Carol Robinson Group
Last NameWu
First NameDi
AddressKavli Institute for Nanoscience Discovery, University of Oxford, Oxford, OX1 3QU, UK
Emaildi.wu2@chem.ox.ac.uk
Phone+4401865275278
Submit Date2023-09-14
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2024-03-14
Release Version1
Di Wu Di Wu
https://dx.doi.org/10.21228/M8F422
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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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

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Treatment
SA314465LAT1-4F2hc_WT_delipidated_2Delipidated
SA314466LAT1-4F2hc_WT_delipidated_1Delipidated
SA314467LAT1-4F2hc_WT_2Untreated
SA314468LAT1-4F2hc_WT_1Untreated
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
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