#METABOLOMICS WORKBENCH huangsxtcm_20230810_063020 DATATRACK_ID:4214 STUDY_ID:ST002848 ANALYSIS_ID:AN004666 PROJECT_ID:PR001783 VERSION 1 CREATED_ON September 6, 2023, 5:47 pm #PROJECT PR:PROJECT_TITLE Lipidomes for linked 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 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 PR:PROJECT_SUMMARY nearly universally size mismatch with its ligands, which results from 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 provide 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 Lipidomes for linked 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 Buffer-1 Buffer-1 Experimental factors:Buffer control RAW_FILE_NAME=072712SX03_NP0725121.mzdata SUBJECT_SAMPLE_FACTORS Buffer-2 Buffer-2 Experimental factors:Buffer control RAW_FILE_NAME=072712SX04_NP0725122.mzdata SUBJECT_SAMPLE_FACTORS Buffer-3 Buffer-3 Experimental factors:Buffer control RAW_FILE_NAME=072712SX05_NP0725123.mzdata SUBJECT_SAMPLE_FACTORS K562CD1a-1 K562CD1a-1 Experimental factors:CD1a RAW_FILE_NAME=072712SX06_K562CD1a061112-0725121.mzdata SUBJECT_SAMPLE_FACTORS K562CD1a-2 K562CD1a-2 Experimental factors:CD1a RAW_FILE_NAME=072712SX07_K562CD1a061112-0725122.mzdata SUBJECT_SAMPLE_FACTORS K562CD1a-3 K562CD1a-3 Experimental factors:CD1a RAW_FILE_NAME=072712SX08_K562CD1a061112-0725123.mzdata SUBJECT_SAMPLE_FACTORS K562CD1b-1 K562CD1b-1 Experimental factors:CD1b RAW_FILE_NAME=072712SX09_K562CD1b042012-0725121.mzdata SUBJECT_SAMPLE_FACTORS K562CD1b-2 K562CD1b-2 Experimental factors:CD1b RAW_FILE_NAME=072712SX10_K562CD1b042012-0725122.mzdata SUBJECT_SAMPLE_FACTORS K562CD1b-3 K562CD1b-3 Experimental factors:CD1b RAW_FILE_NAME=072712SX11_K562CD1b042012-0725123.mzdata SUBJECT_SAMPLE_FACTORS K562CD1c-1 K562CD1c-1 Experimental factors:CD1c RAW_FILE_NAME=072712SX13_K562CD1c070112-0725121.mzdata SUBJECT_SAMPLE_FACTORS K562CD1c-2 K562CD1c-2 Experimental factors:CD1c RAW_FILE_NAME=072712SX14_K562CD1c070112-0725122.mzdata SUBJECT_SAMPLE_FACTORS K562CD1c-3 K562CD1c-3 Experimental factors:CD1c RAW_FILE_NAME=072712SX15_K562CD1c070112-0725123.mzdata SUBJECT_SAMPLE_FACTORS K562CD1d-1 K562CD1d-1 Experimental factors:CD1d RAW_FILE_NAME=072712SX16_K562CD1d072512-0725121.mzdata SUBJECT_SAMPLE_FACTORS K562CD1d-2 K562CD1d-2 Experimental factors:CD1d RAW_FILE_NAME=072712SX17_K562CD1d072512-0725122.mzdata SUBJECT_SAMPLE_FACTORS K562CD1d-3 K562CD1d-3 Experimental factors:CD1d RAW_FILE_NAME=072712SX18_K562CD1d072512-0725123.mzdata SUBJECT_SAMPLE_FACTORS K562B27-1 K562B27-1 Experimental factors:B27 control RAW_FILE_NAME=072712SX19_K562B27051612-0725121.mzdata SUBJECT_SAMPLE_FACTORS K562B27-2 K562B27-2 Experimental factors:B27 control RAW_FILE_NAME=072712SX20_K562B27051612-0725122.mzdata SUBJECT_SAMPLE_FACTORS K562B27-3 K562B27-3 Experimental factors:B27 control RAW_FILE_NAME=072712SX21_K562B27051612-0725123.mzdata #COLLECTION CO:COLLECTION_SUMMARY Lipidomic profiles were generated from lipids that were extracted from K562 CO:COLLECTION_SUMMARY expressed CD1a, CD1b, CD1c, and CD1d proteins compared with peptide-binding CO:COLLECTION_SUMMARY protein HLA-B27 and buffer-only controls. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY Lipidomic profiles were generated from lipids that were extracted from K562 TR:TREATMENT_SUMMARY expressed CD1a, CD1b, CD1c, and CD1d proteins compared with peptide-binding TR:TREATMENT_SUMMARY protein HLA-B27 and buffer-only controls. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Lipid extraction. CD1 and MHC proteins were extracted in chloroform: methanol: SP:SAMPLEPREP_SUMMARY water (2:2:1) (V: V: V) (Figure S1) at room temperature for 30 mins and SP:SAMPLEPREP_SUMMARY centrifuged at 500g for 10 mins. The aqueous phase contained few lipids, so the SP:SAMPLEPREP_SUMMARY combined interphase and organic phase were transferred and stored at -20°C for SP:SAMPLEPREP_SUMMARY comparative lipidomics analysis, which was conducted in parallel with groups of SP:SAMPLEPREP_SUMMARY lipid extracts. Mass spectrometry-based comparative lipidomics. Lipid eluents SP:SAMPLEPREP_SUMMARY were annormalized to protein mass (20-80 μg), dried under nitrogen at 20°C, SP:SAMPLEPREP_SUMMARY dissolved and briefly sonicated in starting mobile phase, which was 100% solvent SP:SAMPLEPREP_SUMMARY B containing 70% hexanes, 30% isopropanol, 0.1% formic acid, and 0.05% ammonium SP:SAMPLEPREP_SUMMARY hydroxide. Triplicate samples for each protein analyzed with blanks that were SP:SAMPLEPREP_SUMMARY intermixed and monitored for lipid carryover, using an Agilent 1200 series HPLC SP:SAMPLEPREP_SUMMARY autosampler with an Agilent 6520 Accurate-Mass Q-TOF MS controlled by MassHunter SP:SAMPLEPREP_SUMMARY software. A normal phase gradient with solvent A (70% isopropanol, 30% methanol, SP:SAMPLEPREP_SUMMARY 0.1% formic acid, and 0.05% ammonium hydroxide) and solvent B through a SP:SAMPLEPREP_SUMMARY MonoChrom Diol column (3 μm X 150 mm X 2 mm; Varian, A0542150X020) were SP:SAMPLEPREP_SUMMARY connected to a MetaGuard guard column (2 mm, Varian, A0542-MG2). The binary SP:SAMPLEPREP_SUMMARY gradient was monitored with solvent B with 100% at 0-10 min, 50% at 17-22 min, SP:SAMPLEPREP_SUMMARY 0% at 30-35 min, and 100% at 40-50 min, followed by an additional 0% for 6 min SP:SAMPLEPREP_SUMMARY post-run for regeneration. Ionization occurred with a dual-electrospray SP:SAMPLEPREP_SUMMARY ionization source maintained at 325°C with a drying gas flow of 5 L/min, SP:SAMPLEPREP_SUMMARY nebulizer pressure of 30 pounds per square inch, and a capillary voltage of 5.5 SP:SAMPLEPREP_SUMMARY kV. Positive- and negative-ion modes were typically monitored between m/z SP:SAMPLEPREP_SUMMARY 100-3000 with the acquisition rate of 1.4 spectra/sec and 713.7 ms/spectrum. SP:SAMPLEPREP_SUMMARY Internal calibrants (Agilent G1969-85001, m/z 121.050573, 922.009798) were SP:SAMPLEPREP_SUMMARY continuously monitored to assess electrospray efficiency and mass accuracy. SP:SAMPLEPREP_SUMMARY NanoESI-CID-MS was typically performed at a collision energy of 35V and an SP:SAMPLEPREP_SUMMARY isolation width of 1.3 m/z and adjusted to optimize signal during individual SP:SAMPLEPREP_SUMMARY experiments. For the semi-quantitative analysis of PCs and SMs eluted from SP:SAMPLEPREP_SUMMARY cleavable CD1, the lipid eluents were normalized based on input protein and 10 SP:SAMPLEPREP_SUMMARY µl were injected into a reverse-phase HPLC-MS system (Agilent Poroshell EC-C18 SP:SAMPLEPREP_SUMMARY column, 1.9-micron, 3 x 50 mm with an Agilent 6520 QTOF mass spectrometry).(van SP:SAMPLEPREP_SUMMARY 't Klooster et al., 2020) #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 MonoChrom Diol column (3 μm X 150 mm X 2 mm; Varian, A0542150X020) 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 ST002848_AN004666_Results.txt UNITS:542 Has m/z:Yes Has RT:Yes RT units:Minutes #END