#METABOLOMICS WORKBENCH huangsxtcm_20230810_100458 DATATRACK_ID:4217 STUDY_ID:ST002849 ANALYSIS_ID:AN004667 PROJECT_ID:PR001783 VERSION 1 CREATED_ON September 6, 2023, 6:15 pm #PROJECT PR:PROJECT_TITLE Lipidomics for unlinked CD1 proteins PR:PROJECT_SUMMARY The cellular CD1 system binds lipid antigens for display to T cells. Here we PR:PROJECT_SUMMARY developed a lipidomics platform that detected > 2000 distinct lipids in cellular PR:PROJECT_SUMMARY CD1 complexes, demonstrating a broad display of self-sphingolipids and PR:PROJECT_SUMMARY phospholipids to T cells. The four types of human CD1 antigen presenting PR:PROJECT_SUMMARY molecules show differing lipid capture motifs based on the length and chemical PR:PROJECT_SUMMARY structures of lipids bound, pointing to general self-lipid capture mechanisms. PR:PROJECT_SUMMARY For CD1a and CD1d, lipid size matches CD1 cleft volume, whereas CD1b shows a PR:PROJECT_SUMMARY nearly universal size mismatch with its ligands, which results from the uniform PR:PROJECT_SUMMARY seating of two small lipids within its large cleft. Further, the list of PR:PROJECT_SUMMARY compounds that comprise the assembled CD1 lipidomes provides a resource for PR:PROJECT_SUMMARY matching to bioactive lipids from other fields of research and supports the PR:PROJECT_SUMMARY ongoing discovery of lipid blockers and antigens for T cells. PR:INSTITUTE Brigham and Women's Hospital PR:LAST_NAME Huang PR:FIRST_NAME Shouxiong PR:ADDRESS 251 Kettering Laboratory Complex, 160 Panzeca Way, Cincinnati, OH PR:EMAIL shouxiong.huang@uc.edu PR:PHONE 5135587572 #STUDY ST:STUDY_TITLE Lipidomics for unlinked CD1 proteins ST:STUDY_SUMMARY The cellular CD1 system binds lipid antigens for display to T cells. Here we ST:STUDY_SUMMARY developed a lipidomics platform that detected > 2000 distinct lipids in cellular ST:STUDY_SUMMARY CD1 complexes, demonstrating a broad display of self-sphingolipids and ST:STUDY_SUMMARY phospholipids to T cells. The four types of human CD1 antigen presenting ST:STUDY_SUMMARY molecules show differing lipid capture motifs based on the length and chemical ST:STUDY_SUMMARY structures of lipids bound, pointing to general self-lipid capture mechanisms. ST:STUDY_SUMMARY For CD1a and CD1d, lipid size matches CD1 cleft volume, whereas CD1b shows a ST:STUDY_SUMMARY nearly universal size mismatch with its ligands, which results from the uniform ST:STUDY_SUMMARY seating of two small lipids within its large cleft. Further, the list of ST:STUDY_SUMMARY compounds that comprise the assembled CD1 lipidomes provides a resource for ST:STUDY_SUMMARY matching to bioactive lipids from other fields of research and supports the ST:STUDY_SUMMARY ongoing discovery of lipid blockers and antigens for T cells. ST:INSTITUTE Brigham and Women's Hospital ST:LAST_NAME Huang ST:FIRST_NAME Shouxiong ST:ADDRESS 251 Kettering Laboratory Complex, 160 Panzeca Way, Cincinnati, OH ST:EMAIL shouxiong.huang@uc.edu ST:PHONE 5135587572 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - H2O-1 Experimental factors:Blank control RAW_FILE_NAME=020711SX02-H2O1.mzdata SUBJECT_SAMPLE_FACTORS - H2O-2 Experimental factors:Blank control RAW_FILE_NAME=020711SX03-H2O2.mzdata SUBJECT_SAMPLE_FACTORS - H2O-3 Experimental factors:Blank control RAW_FILE_NAME=020711SX04-H2O3.mzdata SUBJECT_SAMPLE_FACTORS - NIHCD1a-1 Experimental factors:CD1a RAW_FILE_NAME=020711SX05-NIHCD1a1.mzdata SUBJECT_SAMPLE_FACTORS - NIHCD1a-2 Experimental factors:CD1a RAW_FILE_NAME=020711SX06-NIHCD1a2.mzdata SUBJECT_SAMPLE_FACTORS - NIHCD1a-3 Experimental factors:CD1a RAW_FILE_NAME=020711SX07-NIHCD1a3.mzdata SUBJECT_SAMPLE_FACTORS - NIHCD1b-1 Experimental factors:CD1b RAW_FILE_NAME=020711SX09-NIHCD1b1.mzdata SUBJECT_SAMPLE_FACTORS - NIHCD1b-2 Experimental factors:CD1b RAW_FILE_NAME=020711SX10-NIHCD1b2.mzdata SUBJECT_SAMPLE_FACTORS - NIHCD1b-3 Experimental factors:CD1b RAW_FILE_NAME=020711SX11-NIHCD1b3.mzdata SUBJECT_SAMPLE_FACTORS - NIHCD1c-1 Experimental factors:CD1c RAW_FILE_NAME=020711SX12-NIHCD1c1.mzdata SUBJECT_SAMPLE_FACTORS - NIHCD1c-2 Experimental factors:CD1c RAW_FILE_NAME=020711SX13-NIHCD1c2.mzdata SUBJECT_SAMPLE_FACTORS - NIHCD1c-3 Experimental factors:CD1c RAW_FILE_NAME=020711SX14-NIHCD1c3.mzdata SUBJECT_SAMPLE_FACTORS - NIHCD1d-1 Experimental factors:CD1d RAW_FILE_NAME=020711SX16-NIHCD1d1.mzdata SUBJECT_SAMPLE_FACTORS - NIHCD1d-2 Experimental factors:CD1d RAW_FILE_NAME=020711SX17-NIHCD1d2.mzdata SUBJECT_SAMPLE_FACTORS - NIHCD1d-3 Experimental factors:CD1d RAW_FILE_NAME=020711SX18-NIHCD1d3.mzdata SUBJECT_SAMPLE_FACTORS - NIHMamu-1 Experimental factors:Mamu control RAW_FILE_NAME=020711SX19-Mamu1.mzdata SUBJECT_SAMPLE_FACTORS - NIHMamu-2 Experimental factors:Mamu control RAW_FILE_NAME=020711SX20-Mamu2.mzdata SUBJECT_SAMPLE_FACTORS - NIHMamu-3 Experimental factors:Mamu control RAW_FILE_NAME=020711SX21-Mamu3.mzdata #COLLECTION CO:COLLECTION_SUMMARY Lipidomic profiles were generated from lipids that were extracted from CO:COLLECTION_SUMMARY HEK293T-expressed CD1a, CD1b, CD1c, and CD1d proteins compared with CO:COLLECTION_SUMMARY peptide-binding protein MamuA01 and H2O blank controls. CO:SAMPLE_TYPE HEK cells #TREATMENT TR:TREATMENT_SUMMARY Lipidomic profiles were generated from lipids that were extracted from TR:TREATMENT_SUMMARY HEK293T-expressed CD1a, CD1b, CD1c, and CD1d proteins compared with TR:TREATMENT_SUMMARY peptide-binding protein MamuA01 and H2O blank controls. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Lipidomic profiles were generated from lipids that were extracted from SP:SAMPLEPREP_SUMMARY HEK293T-expressed CD1a, CD1b, CD1c, and CD1d proteins compared with SP:SAMPLEPREP_SUMMARY peptide-binding protein MamuA01 and H2O blank controls. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Triplicate samples for each protein analyzed with blanks that were intermixed CH:CHROMATOGRAPHY_SUMMARY and monitored for lipid carryover, using an Agilent 1200 series HPLC autosampler CH:CHROMATOGRAPHY_SUMMARY with an Agilent 6520 Accurate-Mass Q-TOF MS controlled by MassHunter software. CH:CHROMATOGRAPHY_TYPE Normal phase CH:INSTRUMENT_NAME Agilent 6520 Accurate-Mass Q-TOF CH:COLUMN_NAME a MetaGuard guard column (2 mm, Varian, A0542-MG2) CH:SOLVENT_A 70% isopropanol, 30% methanol, 0.1% formic acid, and 0.05% ammonium hydroxide CH:SOLVENT_B 70% hexanes, 30% isopropanol, 0.1% formic acid, and 0.05% ammonium hydroxide CH:FLOW_GRADIENT The binary gradient was monitored with solvent B with 100% at 0-10 min, 50% at CH:FLOW_GRADIENT 17-22 min, 0% at 30-35 min, and 100% at 40-50 min, followed by an additional 0% CH:FLOW_GRADIENT for 6 min post-run for regeneration. CH:FLOW_RATE 0.7 ml/min CH:COLUMN_TEMPERATURE 22 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6520 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS MS data were acquired with MassHunter software. MS:MS_RESULTS_FILE ST002849_AN004667_Results.txt UNITS:631 Has m/z:Yes Has RT:Yes RT units:Minutes #END