{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002875","ANALYSIS_ID":"AN004712","VERSION":"1","CREATED_ON":"September 25, 2023, 9:28 am"},

"PROJECT":{"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-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","EMAIL":"di.wu2@chem.ox.ac.uk","PHONE":"+4401865275278"},

"STUDY":{"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","EMAIL":"di.wu2@chem.ox.ac.uk","PHONE":"+4401865275278"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606","CELL_STRAIN_DETAILS":"HEK293F"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"LAT1-4F2hc_WT_1",
"Factors":{"Treatment":"Untreated"},
"Additional sample data":{"RAW_FILE_NAME":"LAT1-4F2hc_WT_1.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"LAT1-4F2hc_WT_2",
"Factors":{"Treatment":"Untreated"},
"Additional sample data":{"RAW_FILE_NAME":"LAT1-4F2hc_WT_2.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"LAT1-4F2hc_WT_delipidated_1",
"Factors":{"Treatment":"Delipidated"},
"Additional sample data":{"RAW_FILE_NAME":"LAT1-4F2hc_WT_delipidated_1.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"LAT1-4F2hc_WT_delipidated_2",
"Factors":{"Treatment":"Delipidated"},
"Additional sample data":{"RAW_FILE_NAME":"LAT1-4F2hc_WT_delipidated_2.mzML"}
}
],
"COLLECTION":{"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_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."},

"SAMPLEPREP":{"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."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Column information: https://www.thermofisher.com/order/catalog/product/164568","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Dionex Ultimate 3000","COLUMN_NAME":"Thermo Acclaim PepMap 100 C18 (150 x 0.007 mm, 3um, 100A)","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","FLOW_GRADIENT":"30%-99%","FLOW_RATE":"300 nl/min","COLUMN_TEMPERATURE":"40"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo LTQ XL","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"-","MS_RESULTS_FILE":"ST002875_AN004712_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes"}

}