{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002911","ANALYSIS_ID":"AN004781","VERSION":"1","CREATED_ON":"October 2, 2023, 2:40 pm"},

"PROJECT":{"PROJECT_TITLE":"LiLA: Lipid Lung-based ATLAS built Through a Comprehensive Workflow Designed for an Accurate Lipid Annotation","PROJECT_TYPE":"Untargeted Lipidomics to build a Lung Tissue ATLAS","PROJECT_SUMMARY":"Accurate lipid annotation is crucial for understanding the role of lipids in health and disease and identifying therapeutic targets. However, annotating the wide variety of lipid species in biological samples remains challenging in untargeted lipidomic studies. In this work, we present an optimized lipid annotation workflow based on the combination of LC-MS and MS/MS strategies, four bioinformatic tools, and a decision-tree-based approach to support the accurate annotation and semi-quantification of the lipid species present in lung tissue from control mice. The developed workflow allowed us to generate a lipid lung-based ATLAS (LiLA), which was then employed to unveil the lipidomic signatures of the Mycobacterium tuberculosis (Mtb) infection at two different time points for a deeper understanding of the disease progression. This workflow, combined with manual inspection strategies of MS/MS data, can enhance the annotation process for lipidomic studies and guide the generation of sample-specific lipidome maps. LiLA serves as a freely available data resource that can be employed in future studies to address lipidomic alterations in mice lung tissue.","INSTITUTE":"Universidad CEU San Pablo","DEPARTMENT":"Centro de MEtabolómica y Bioanálisis (CEMBIO)","LAST_NAME":"Gonzalez-Riano","FIRST_NAME":"Carolina","ADDRESS":"Facultad de Farmacia, Universidad CEU San Pablo, Campus Monteprincipe, Boadilla del Monte, Boadilla del Monte, Madrid, 28668, Spain","EMAIL":"car.gonzalez@ceindo.ceu.es","PHONE":"00 34 91 3724753","FUNDING_SOURCE":"This work was supported by grants from the following entities: Ministry of Science and Innovation of Spain (MICINN) and the European Regional Development Fund FEDER, grant number PID2021-122490NB-I00; and National Institute of Allergy and Infectious Diseases (NIAID)-University of Alabama (USA), grant number MUSUAB2."},

"STUDY":{"STUDY_TITLE":"LiLA: Lipid Lung-based ATLAS built Through a Comprehensive Workflow Designed for an Accurate Lipid Annotation","STUDY_SUMMARY":"Accurate lipid annotation is crucial for understanding the role of lipids in health and disease and identifying therapeutic targets. However, annotating the wide variety of lipid species in biological samples remains challenging in untargeted lipidomic studies. In this work, we present an optimized lipid annotation workflow based on the combination of LC-MS and MS/MS strategies, four bioinformatic tools, and a decision-tree-based approach to support the accurate annotation and semi-quantification of the lipid species present in lung tissue from control mice. The developed workflow allowed us to generate a lipid lung-based ATLAS (LiLA), which was then employed to unveil the lipidomic signatures of the Mycobacterium tuberculosis (Mtb) infection at two different time points for a deeper understanding of the disease progression. This workflow, combined with manual inspection strategies of MS/MS data, can enhance the annotation process for lipidomic studies and guide the generation of sample-specific lipidome maps. LiLA serves as a freely available data resource that can be employed in future studies to address lipidomic alterations in mice lung tissue.","INSTITUTE":"Universidad CEU San Pablo","DEPARTMENT":"Centro de MEtabolómica y Bioanálisis (CEMBIO)","LAST_NAME":"González","FIRST_NAME":"Carolina","ADDRESS":"km 0, Universidad CEU-San Pablo Urbanización Montepríncipe. M-501","EMAIL":"carolina.gonzalezriano@ceu.es","PHONE":"646251045"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090","AGE_OR_AGE_RANGE":"Seven- to eight-week-old female BALB/c mice","GENDER":"Female","ANIMAL_ANIMAL_SUPPLIER":"Jackson Laboratories"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"1_UT1",
"Factors":{"Factor":"4-weeks infection"},
"Additional sample data":{"RAW_FILE_NAME":"1_UT1"}
},
{
"Subject ID":"-",
"Sample ID":"1_UT2",
"Factors":{"Factor":"4-weeks infection"},
"Additional sample data":{"RAW_FILE_NAME":"1_UT2"}
},
{
"Subject ID":"-",
"Sample ID":"1_UT3",
"Factors":{"Factor":"4-weeks infection"},
"Additional sample data":{"RAW_FILE_NAME":"1_UT3"}
},
{
"Subject ID":"-",
"Sample ID":"1_UT4",
"Factors":{"Factor":"4-weeks infection"},
"Additional sample data":{"RAW_FILE_NAME":"1_UT4"}
},
{
"Subject ID":"-",
"Sample ID":"1_UT5",
"Factors":{"Factor":"4-weeks infection"},
"Additional sample data":{"RAW_FILE_NAME":"1_UT5"}
},
{
"Subject ID":"-",
"Sample ID":"1_UT6",
"Factors":{"Factor":"4-weeks infection"},
"Additional sample data":{"RAW_FILE_NAME":"1_UT6"}
},
{
"Subject ID":"-",
"Sample ID":"12_UT1",
"Factors":{"Factor":"12-weeks infection"},
"Additional sample data":{"RAW_FILE_NAME":"12_UT1"}
},
{
"Subject ID":"-",
"Sample ID":"12_UT2",
"Factors":{"Factor":"12-weeks infection"},
"Additional sample data":{"RAW_FILE_NAME":"12_UT2"}
},
{
"Subject ID":"-",
"Sample ID":"12_UT3",
"Factors":{"Factor":"12-weeks infection"},
"Additional sample data":{"RAW_FILE_NAME":"12_UT3"}
},
{
"Subject ID":"-",
"Sample ID":"12_UT4",
"Factors":{"Factor":"12-weeks infection"},
"Additional sample data":{"RAW_FILE_NAME":"12_UT4"}
},
{
"Subject ID":"-",
"Sample ID":"12_UT5",
"Factors":{"Factor":"12-weeks infection"},
"Additional sample data":{"RAW_FILE_NAME":"12_UT5"}
},
{
"Subject ID":"-",
"Sample ID":"1_UI1",
"Factors":{"Factor":"control"},
"Additional sample data":{"RAW_FILE_NAME":"1_UI1"}
},
{
"Subject ID":"-",
"Sample ID":"1_UI2",
"Factors":{"Factor":"control"},
"Additional sample data":{"RAW_FILE_NAME":"1_UI2"}
},
{
"Subject ID":"-",
"Sample ID":"1_UI3",
"Factors":{"Factor":"control"},
"Additional sample data":{"RAW_FILE_NAME":"1_UI3"}
},
{
"Subject ID":"-",
"Sample ID":"1_UI4",
"Factors":{"Factor":"control"},
"Additional sample data":{"RAW_FILE_NAME":"1_UI4"}
},
{
"Subject ID":"-",
"Sample ID":"1_UI5",
"Factors":{"Factor":"control"},
"Additional sample data":{"RAW_FILE_NAME":"1_UI5"}
},
{
"Subject ID":"-",
"Sample ID":"1_UI6",
"Factors":{"Factor":"control"},
"Additional sample data":{"RAW_FILE_NAME":"1_UI6"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Animals were divided into three different groups: healthy controls (Mtb-, n=6), infected mice four weeks post-infection (Mtb+4w, n=6) and infected mice twelve weeks post-infection (Mtb+12w, n=5). Seven- to eight-week-old female BALB/c mice were purchased from Jackson Laboratories. Mice were housed in a pathogen-free facility with ad libitum access to water and food. All the animal studies were performed following the guidelines approved by IACUC at the University of Alabama at Birmingham, USA. Mice were aerosol infected with Mtb H37Rv using the aerosol inhalation exposure system (Glas-Col, USA) to deliver ~120-250 CFU/mouse lung. The infection dose was estimated by enumerating the lung CFU at 24 hours post-infection. Mice were sacrificed using anaesthesia with isoflurane followed by gentle cervical dislocation. Mice organs were aseptically harvested and homogenized in 2 ml of 1x PBS, pH 7.4. Serial dilutions of homogenates were prepared in 1x PBS and plated on 7H11 agar plates supplemented with 10% ADS (Albumin, Dextrose, and NaCl), Carbenicillin (25 mg/L) and Cycloheximide (25 mg/L). Plates were incubated at 37 °C for ~21 days before counting colonies. Finally, lung samples were transferred to an N2(l)-containing recipient to freeze the tissues and stored at -80 °C to avoid postmortem metabolic processes.","SAMPLE_TYPE":"Lung"},

"TREATMENT":{"TREATMENT_SUMMARY":"Animals were divided into three different groups: healthy controls (Mtb-, n=6), infected mice four weeks post-infection (Mtb+4w, n=6) and infected mice twelve weeks post-infection (Mtb+12w, n=5). Seven- to eight-week-old female BALB/c mice were purchased from Jackson Laboratories. Mice were housed in a pathogen-free facility with ad libitum access to water and food. All the animal studies were performed following the guidelines approved by IACUC at the University of Alabama at Birmingham, USA. Mice were aerosol infected with Mtb H37Rv using the aerosol inhalation exposure system (Glas-Col, USA) to deliver ~120-250 CFU/mouse lung. The infection dose was estimated by enumerating the lung CFU at 24 hours post-infection. Mice were sacrificed using anaesthesia with isoflurane followed by gentle cervical dislocation. Mice organs were aseptically harvested and homogenized in 2 ml of 1x PBS, pH 7.4. Serial dilutions of homogenates were prepared in 1x PBS and plated on 7H11 agar plates supplemented with 10% ADS (Albumin, Dextrose, and NaCl), Carbenicillin (25 mg/L) and Cycloheximide (25 mg/L). Plates were incubated at 37 °C for ~21 days before counting colonies. Finally, lung samples were transferred to an N2(l)-containing recipient to freeze the tissues and stored at -80 °C to avoid postmortem metabolic processes."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"The sample preparation and lipid extraction were performed at the Centers for AIDS Research and Free Radical Biology, University of Alabama at Birmingham (Birmingham, AL, United States), following a protocol initially described and optimized at CEMBIO (Madrid, Spain). Approximately 75 mg of lung tissue was mixed with a cold (–20°C) mixture of MeOH:H2O (1:1, v/v) added in a ratio of 1 mg tissue:10 µL of extraction solvent. Next, the tissue samples were homogenized using the Dounce homogenizer. After the homogenization, 200 µL of homogenate was mixed with 640 µL of MeOH and 160 µL of Methyl-Tert-Butyl ether (MTBE) to extract hydrophobic compounds. Samples were then vortex-mixed for 1 hour at room temperature (RT) and centrifuged at 4000 g for 20 min at 20°C. The samples were then passed through spin X columns (0.22 µm filter), and 200 µL of the filtered sample was dried at RT in the vacuum concentrator. From here, the samples were sent to CEMBIO for the UHPLC-MS analysis. Prior to the analysis, dried samples were re-suspended with 200 µL of MeOH/MTBE/H2O (7.4:1.6:1, v/v/v), which contained the corresponding ISs (C17 – sphingosine at 1 ppm for positive ion mode, and d31–palmitic acid at 3 ppm for negative ion mode). Samples were then centrifuged (16,100xg, 5 min, 15°C) before transferring them into sample vials with glass inserts for LC-MS analysis."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Agilent 1290 Infinity II","COLUMN_NAME":"Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um)","SOLVENT_A":"90% water/10% methanol; 10 mM ammonium acetate; 0.2 mM ammonium fluoride","SOLVENT_B":"20% acetonitrile/30% methanol/50% isopropanol; 10 mM ammonium acetate; 0.2 mM ammonium fluoride","FLOW_GRADIENT":"Started at 70% of B at 0 –1 min, 86% at 3.5 –10 min, and 100% B at 11–17 min","FLOW_RATE":"0.6 mL/min","COLUMN_TEMPERATURE":"50 °C"},

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

"MS":{"INSTRUMENT_NAME":"Agilent 6545 QTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"150 V fragmentor, 65 V skimmer, 3500 V capillary voltage, 750 V octopole radio frequency voltage, 10 L/min nebulizer gas flow, 200 °C gas temperature, 50 psi nebulizer gas pressure, 12 L/min sheath gas flow, and 300 °C sheath gas temperature. Data were collected in positive and negative ESI modes in separate runs, operated in full scan mode from 40 to 1700 m/z with a scan rate of 3 spectra/s. A solution consisting of two reference mass compounds was infused throughout the whole analysis: purine (C5H4N4) at m/z 119.0363 and HP-0921 (C18H18O6N3P3F24) at m/z 980.0163 (HP-0921 + acetate). These masses were continuously infused into the system through an Agilent 1260 Iso Pump at a 1 mL/min (split ratio 1:100) to provide a constant mass correction."},

"MS_METABOLITE_DATA":{
"Units":"AREA",

"Data":[{"Metabolite":"BMP 16:0_16:0","1_UI1":"76727.10544","1_UI2":"61618.65818","1_UI3":"66519.11629","1_UI4":"67531.84547","1_UI5":"74303.50229","1_UI6":"66716.66483","1_UT1":"69598.99949","1_UT2":"90511.47336","1_UT3":"87864.21646","1_UT4":"73678.26446","1_UT5":"76877.73105","1_UT6":"90791.70926","12_UT1":"88385.63234","12_UT2":"87744.71438","12_UT3":"114581.3514","12_UT4":"115986.4905","12_UT5":"111567.075"},{"Metabolite":"BMP 16:0_20:4","1_UI1":"42185.10234","1_UI2":"37354.44071","1_UI3":"37304.48469","1_UI4":"39751.39474","1_UI5":"43224.34756","1_UI6":"39363.9422","1_UT1":"44253.555","1_UT2":"57050.04693","1_UT3":"65774.86978","1_UT4":"55465.70611","1_UT5":"57539.92837","1_UT6":"51102.12445","12_UT1":"64489.99165","12_UT2":"66407.48875","12_UT3":"88927.07685","12_UT4":"85965.10617","12_UT5":"86285.99884"},{"Metabolite":"BMP 18:1_22:6","1_UI1":"13527.71036","1_UI2":"11078.22649","1_UI3":"11268.26209","1_UI4":"13263.58016","1_UI5":"12346.54284","1_UI6":"12224.35599","1_UT1":"14455.66831","1_UT2":"20598.53495","1_UT3":"22539.50422","1_UT4":"20110.94585","1_UT5":"18146.77038","1_UT6":"15553.62905","12_UT1":"21367.5567","12_UT2":"26124.15127","12_UT3":"33494.26996","12_UT4":"33658.63407","12_UT5":"32926.90518"},{"Metabolite":"BMP 18:2_22:6","1_UI1":"12022.61034","1_UI2":"9318.11195","1_UI3":"9436.912469","1_UI4":"10460.62325","1_UI5":"10824.97014","1_UI6":"10040.08563","1_UT1":"15726.83699","1_UT2":"24277.34565","1_UT3":"22960.77578","1_UT4":"23572.7962","1_UT5":"18522.62086","1_UT6":"17018.25561","12_UT1":"15753.83225","12_UT2":"22494.45038","12_UT3":"33312.77505","12_UT4":"27613.72404","12_UT5":"27858.85012"},{"Metabolite":"BMP 20:4_22:6","1_UI1":"3695.490093","1_UI2":"3323.883377","1_UI3":"3187.833498","1_UI4":"3672.626265","1_UI5":"3723.584313","1_UI6":"3715.52702","1_UT1":"6228.684671","1_UT2":"8686.955865","1_UT3":"9449.934891","1_UT4":"8575.431875","1_UT5":"8415.592125","1_UT6":"6018.743673","12_UT1":"11763.16952","12_UT2":"14947.39742","12_UT3":"22011.00958","12_UT4":"21135.06123","12_UT5":"20271.13202"},{"Metabolite":"BMP 22:6_22:6","1_UI1":"17214.10555","1_UI2":"15572.53137","1_UI3":"16101.95413","1_UI4":"22575.26478","1_UI5":"16812.83265","1_UI6":"18468.79948","1_UT1":"27314.83421","1_UT2":"45624.5887","1_UT3":"45313.89159","1_UT4":"40489.37769","1_UT5":"37464.4343","1_UT6":"28227.30883","12_UT1":"28291.43286","12_UT2":"37565.92006","12_UT3":"55173.40909","12_UT4":"56277.34585","12_UT5":"53355.39372"},{"Metabolite":"Cer 16:0;2O/18:1","1_UI1":"136843.9643","1_UI2":"160309.4828","1_UI3":"201985.44","1_UI4":"216106.0967","1_UI5":"202048.4207","1_UI6":"216950.629","1_UT1":"251180.4607","1_UT2":"318659.8772","1_UT3":"304966.0284","1_UT4":"248311.9716","1_UT5":"294128.7208","1_UT6":"335327.039","12_UT1":"251100.962","12_UT2":"318268.6261","12_UT3":"364493.3713","12_UT4":"351992.8738","12_UT5":"342084.5755"},{"Metabolite":"Cer 16:0;2O/18:2","1_UI1":"30049.75756","1_UI2":"51580.32048","1_UI3":"56874.22248","1_UI4":"65317.57793","1_UI5":"59412.37466","1_UI6":"75251.62298","1_UT1":"52283.62061","1_UT2":"64042.80798","1_UT3":"77158.68992","1_UT4":"66379.01095","1_UT5":"71734.24898","1_UT6":"72363.75329","12_UT1":"39469.69808","12_UT2":"51038.54462","12_UT3":"46603.67463","12_UT4":"61735.40166","12_UT5":"65684.70107"},{"Metabolite":"Cer 18:0;2O/16:0","1_UI1":"19497.77122","1_UI2":"19734.27299","1_UI3":"19244.16453","1_UI4":"26760.88263","1_UI5":"25181.37549","1_UI6":"24790.11894","1_UT1":"37380.48752","1_UT2":"50542.9752","1_UT3":"62744.79293","1_UT4":"47862.87558","1_UT5":"44804.13527","1_UT6":"44158.2852","12_UT1":"48725.13547","12_UT2":"67906.39271","12_UT3":"85357.53767","12_UT4":"74283.48736","12_UT5":"57955.29682"},{"Metabolite":"Cer 18:0;2O/18:0","1_UI1":"11414.30966","1_UI2":"12288.22324","1_UI3":"13316.37496","1_UI4":"13030.71321","1_UI5":"14477.08697","1_UI6":"13661.24526","1_UT1":"14309.02722","1_UT2":"16013.93849","1_UT3":"18996.64838","1_UT4":"14391.03524","1_UT5":"15343.762","1_UT6":"15464.75241","12_UT1":"11995.94404","12_UT2":"14050.48951","12_UT3":"13868.59225","12_UT4":"18571.80665","12_UT5":"18007.63582"},{"Metabolite":"Cer 18:0;2O/18:1","1_UI1":"62889.7455","1_UI2":"76226.73424","1_UI3":"91485.87355","1_UI4":"107035.4048","1_UI5":"92676.63702","1_UI6":"98269.70805","1_UT1":"72111.1707","1_UT2":"85976.50308","1_UT3":"102975.1724","1_UT4":"81979.40461","1_UT5":"89244.67844","1_UT6":"92908.96285","12_UT1":"71727.83782","12_UT2":"73566.73582","12_UT3":"78856.7199","12_UT4":"98774.10828","12_UT5":"102721.8107"},{"Metabolite":"Cer 18:0;2O/20:0","1_UI1":"6667.406949","1_UI2":"7390.731871","1_UI3":"8260.136467","1_UI4":"7946.130538","1_UI5":"8577.809902","1_UI6":"7573.360928","1_UT1":"9363.03304","1_UT2":"10155.45942","1_UT3":"11372.85611","1_UT4":"9483.275629","1_UT5":"8247.915092","1_UT6":"8937.400784","12_UT1":"8521.498042","12_UT2":"8472.149408","12_UT3":"9021.905329","12_UT4":"9880.963114","12_UT5":"9803.115006"},{"Metabolite":"Cer 18:0;2O/22:0","1_UI1":"17548.29021","1_UI2":"21060.80901","1_UI3":"25654.74497","1_UI4":"34094.16036","1_UI5":"26205.02951","1_UI6":"25901.36513","1_UT1":"38688.10699","1_UT2":"47711.47767","1_UT3":"40190.3186","1_UT4":"35127.28374","1_UT5":"33381.08262","1_UT6":"35051.34843","12_UT1":"32721.35681","12_UT2":"31503.67679","12_UT3":"33829.06437","12_UT4":"37618.49781","12_UT5":"44876.42399"},{"Metabolite":"Cer 18:0;2O/23:0","1_UI1":"10146.31154","1_UI2":"12553.22433","1_UI3":"16128.29986","1_UI4":"20036.60865","1_UI5":"15409.58786","1_UI6":"14969.0002","1_UT1":"19101.67674","1_UT2":"23236.05882","1_UT3":"18776.31366","1_UT4":"20066.19299","1_UT5":"13803.58496","1_UT6":"17208.11861","12_UT1":"17797.49931","12_UT2":"14981.35179","12_UT3":"16339.13692","12_UT4":"15475.08601","12_UT5":"21306.68154"},{"Metabolite":"Cer 18:0;2O/24:0","1_UI1":"43538.55021","1_UI2":"58857.7928","1_UI3":"77764.24554","1_UI4":"117049.1116","1_UI5":"69988.27848","1_UI6":"74720.40152","1_UT1":"126864.2271","1_UT2":"160894.9975","1_UT3":"121141.5068","1_UT4":"112150.6699","1_UT5":"101926.6223","1_UT6":"100142.5702","12_UT1":"117450.0123","12_UT2":"104099.8301","12_UT3":"121218.5514","12_UT4":"127617.6295","12_UT5":"161892.4308"},{"Metabolite":"Cer 18:0;2O/24:1","1_UI1":"26480.96152","1_UI2":"50655.65906","1_UI3":"63461.29791","1_UI4":"89699.11194","1_UI5":"64194.70482","1_UI6":"66489.49207","1_UT1":"117251.0663","1_UT2":"155247.106","1_UT3":"124892.2574","1_UT4":"110325.9081","1_UT5":"107462.1534","1_UT6":"112496.2076","12_UT1":"124776.0499","12_UT2":"142332.0246","12_UT3":"175868.5701","12_UT4":"156207.0895","12_UT5":"159372.7678"},{"Metabolite":"Cer 18:0;2O/25:0","1_UI1":"4896.862789","1_UI2":"5977.392695","1_UI3":"7518.600515","1_UI4":"8859.422345","1_UI5":"6471.01694","1_UI6":"7086.191978","1_UT1":"8175.659256","1_UT2":"9109.857271","1_UT3":"7388.90913","1_UT4":"7613.554714","1_UT5":"6381.577766","1_UT6":"6680.669243","12_UT1":"8008.885288","12_UT2":"6860.277764","12_UT3":"7214.057272","12_UT4":"6703.225434","12_UT5":"9084.019673"},{"Metabolite":"Cer 18:0;2O/25:1","1_UI1":"28613.82107","1_UI2":"25395.71966","1_UI3":"29368.85052","1_UI4":"31170.81401","1_UI5":"34192.05581","1_UI6":"27882.0006","1_UT1":"30470.75977","1_UT2":"34104.84071","1_UT3":"37379.73309","1_UT4":"38221.62442","1_UT5":"28207.83021","1_UT6":"34879.86624","12_UT1":"31058.65134","12_UT2":"30407.31364","12_UT3":"36258.25577","12_UT4":"35736.61733","12_UT5":"39407.25128"},{"Metabolite":"Cer 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