#METABOLOMICS WORKBENCH mdaly92_20220511_031454 DATATRACK_ID:3238 STUDY_ID:ST002190 ANALYSIS_ID:AN003584 VERSION 1 CREATED_ON 02-08-2024 #PROJECT PR:PROJECT_TITLE Mass Spectrometry Imaging of Lipids In A Gut Epithelial Cell Model PR:PROJECT_TYPE MS imaging of cells PR:PROJECT_SUMMARY Mass spectrometry imaging of lipids in a gut epithelial cell model PR:INSTITUTE University of Manchester PR:LAST_NAME Mattar PR:FIRST_NAME Hadeer PR:ADDRESS Manchester Institute of Biotechnology, Princess Street, Manchester, UK, M1 7DN PR:EMAIL hmatar@bu.edu.sa PR:PHONE 0161 306 6000 PR:DOI http://dx.doi.org/10.21228/M88H8K #STUDY ST:STUDY_TITLE Mass Spectrometry Imaging of Lipids In A Gut Epithelial Cell Model ST:STUDY_TYPE Mass spectromery imaging of cells. ST:STUDY_SUMMARY Scope: The Caco2/HT29-MTX co-culture system is widely used as a cell model of ST:STUDY_SUMMARY the intestinal epithelium. Although the gut epithelium plays an important role ST:STUDY_SUMMARY in the uptake of free fatty acids and the resynthesis of triglycerides the lipid ST:STUDY_SUMMARY distribution profile of the co-culture system is not well understood. Desorption ST:STUDY_SUMMARY electrospray ionization (DESI) is a mass spectrometry (MS) technique which has ST:STUDY_SUMMARY been widely used to study the main classes of lipid molecules on different ST:STUDY_SUMMARY tissue surfaces. This has been used to map lipid species and their distribution ST:STUDY_SUMMARY in Caco2 and HT29-MTX co-culture system. Methods and results: Caco2 and HT29-MTX ST:STUDY_SUMMARY cells were seeded on coverslips either singly or as cocultures in ratios of ST:STUDY_SUMMARY 75:25 and 50:50. Cells were cultured for 21 days before MS imaging using a DESI ST:STUDY_SUMMARY source in both the positive and negative ionization modes. The identity of ST:STUDY_SUMMARY selected lipids was confirmed in negative and positive ionisation modes using ST:STUDY_SUMMARY tandem MS. Although many lipids were common to both cell lines, there were ST:STUDY_SUMMARY distinctive patterns in the lipidomes. Thus, the lipidome of Caco2 cells was ST:STUDY_SUMMARY more heterogeneous and rich in cholesterol esters and triglycerides whilst ST:STUDY_SUMMARY HT29-MTX cells has a distinctive lipidome relating to phosphatidylethanolamines, ST:STUDY_SUMMARY phosphatidylinositols and odd chain lipids, including C17 fatty acids. ST:STUDY_SUMMARY Conclusion: DESI-MSI has shown that Caco2 and HT29-MTX cells have distinctive ST:STUDY_SUMMARY lipidomes which are still evident when the cells are cocultured. It has ST:STUDY_SUMMARY potential to both allow further validation of these widely used cell models and ST:STUDY_SUMMARY provide insights into how dietary components may modify lipid metabolism in ST:STUDY_SUMMARY future. ST:INSTITUTE Manchester Institute of Biotechnology, University of Manchester ST:LAST_NAME Mattar ST:FIRST_NAME Hadeer ST:ADDRESS Manchester Institute of Biotechnology, Princess Street, Manchester, UK, M1 7DN ST:EMAIL hmatar@bu.edu.sa ST:PHONE 0161 306 6000 ST:SUBMIT_DATE 2022-05-11 #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]Additional sample data SUBJECT_SAMPLE_FACTORS - 06DEC2018_Hadeer_neg_cell5_MS01 Analyte 3 Caco2:0;100 | Ionisation_mode:Negative RAW_FILE_NAME=06DEC2018_Hadeer_neg_cell5_MS01 Analyte 3.mzML SUBJECT_SAMPLE_FACTORS - 06Decv18_manchesterUni_Hadeer_cell5_PosMS01 Analyte 1 Caco2:0;100 | Ionisation_mode:Positive RAW_FILE_NAME=06Decv18_manchesterUni_Hadeer_cell5_PosMS01 Analyte 1.mzML SUBJECT_SAMPLE_FACTORS - 06DEC2018_Hadeer_neg_cell1_MS01 Analyte 3 Caco2:100;0 | Ionisation_mode:Negative RAW_FILE_NAME=06DEC2018_Hadeer_neg_cell1_MS01 Analyte 3.mzML SUBJECT_SAMPLE_FACTORS - 06Decv18_manchesterUni_Hadeer_cell1_PosMS01 Analyte 1 Caco2:100;0 | Ionisation_mode:Positive RAW_FILE_NAME=06Decv18_manchesterUni_Hadeer_cell1_PosMS01 Analyte 1.mzML SUBJECT_SAMPLE_FACTORS - 06DEC2018_Hadeer_neg_cell4_MS01 Analyte 3 Caco2:25;75 | Ionisation_mode:Negative RAW_FILE_NAME=06DEC2018_Hadeer_neg_cell4_MS01 Analyte 3.mzML SUBJECT_SAMPLE_FACTORS - 06Decv18_manchesterUni_Hadeer_cell4_PosMS01 Analyte 1 Caco2:25;75 | Ionisation_mode:Positive RAW_FILE_NAME=06Decv18_manchesterUni_Hadeer_cell4_PosMS01 Analyte 1.mzML SUBJECT_SAMPLE_FACTORS - 06DEC2018_Hadeer_neg_cell3_MS01 Analyte 3 Caco2:50;50 | Ionisation_mode:Negative RAW_FILE_NAME=06DEC2018_Hadeer_neg_cell3_MS01 Analyte 3.mzML SUBJECT_SAMPLE_FACTORS - 06Decv18_manchesterUni_Hadeer_cell3_PosMS01 Analyte 1 Caco2:50;50 | Ionisation_mode:Positive RAW_FILE_NAME=06Decv18_manchesterUni_Hadeer_cell3_PosMS01 Analyte 1.mzML SUBJECT_SAMPLE_FACTORS - 06DEC2018_Hadeer_neg_cell2_MS01 Analyte 3 Caco2:75;25 | Ionisation_mode:Negative RAW_FILE_NAME=06DEC2018_Hadeer_neg_cell2_MS01 Analyte 3.mzML SUBJECT_SAMPLE_FACTORS - 06Decv18_manchesterUni_Hadeer_cell2_PosMS01 Analyte 1 Caco2:75;25 | Ionisation_mode:Positive RAW_FILE_NAME=06Decv18_manchesterUni_Hadeer_cell2_PosMS01 Analyte 1.mzML #COLLECTION CO:COLLECTION_SUMMARY Caco2 and HT29-MTX cells were cultured separately in bulk in 25 cm2 flasks at 37 CO:COLLECTION_SUMMARY ºC, 5% CO2 for 2-3 days. DMEM complete media supplemented with 20% (v/v) foetal CO:COLLECTION_SUMMARY serum bovine, 1% (w/v) non-essential amino acids, 1% (w/v) L-glutamine and 1% CO:COLLECTION_SUMMARY (w/v) penicillin-streptomycin was used as culture medium. On reaching 80–90% CO:COLLECTION_SUMMARY confluency cells were trypsinised and then seeded on a coverslip at a density of CO:COLLECTION_SUMMARY 1x105 cells/ml either as separate cultures or in a co-culture system seeded at CO:COLLECTION_SUMMARY ratios of 75:25, 50:50 and 25:75 (Caco2: HT29-MTXcells). After seeding, each CO:COLLECTION_SUMMARY coverslip was placed in the well of a 6-well cell culture plate, and incubated CO:COLLECTION_SUMMARY at 37 ºC, 5% CO2. Media was changed every 48 h for 21 days until cells were CO:COLLECTION_SUMMARY confluent. Cells were prepared for DESI MSI analysis by rinsing coverslips in CO:COLLECTION_SUMMARY 150 mM ammonium acetate, pH 7.1 for 30 s before being allowed to dry in the air CO:COLLECTION_SUMMARY stream of a biological safety cabinet for 15 min. Coverslips were then CO:COLLECTION_SUMMARY thoroughly dried using a vacuum desiccator for 15 min before storage at -80 °C CO:COLLECTION_SUMMARY in petri dishes until required. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY Caco2 and HT29-MTX cells were cultured separately in bulk in 25 cm2 flasks at 37 TR:TREATMENT_SUMMARY ºC, 5% CO2 for 2-3 days. DMEM complete media supplemented with 20% (v/v) foetal TR:TREATMENT_SUMMARY serum bovine, 1% (w/v) non-essential amino acids, 1% (w/v) L-glutamine and 1% TR:TREATMENT_SUMMARY (w/v) penicillin-streptomycin was used as culture medium. On reaching 80–90% TR:TREATMENT_SUMMARY confluency cells were trypsinised and then seeded on a coverslip at a density of TR:TREATMENT_SUMMARY 1x105 cells/ml either as separate cultures or in a co-culture system seeded at TR:TREATMENT_SUMMARY ratios of 75:25, 50:50 and 25:75 (Caco2: HT29-MTXcells). After seeding, each TR:TREATMENT_SUMMARY coverslip was placed in the well of a 6-well cell culture plate, and incubated TR:TREATMENT_SUMMARY at 37 ºC, 5% CO2. Media was changed every 48 h for 21 days until cells were TR:TREATMENT_SUMMARY confluent. Cells were prepared for DESI MSI analysis by rinsing coverslips in TR:TREATMENT_SUMMARY 150 mM ammonium acetate, pH 7.1 for 30 s before being allowed to dry in the air TR:TREATMENT_SUMMARY stream of a biological safety cabinet for 15 min. Coverslips were then TR:TREATMENT_SUMMARY thoroughly dried using a vacuum desiccator for 15 min before storage at -80 °C TR:TREATMENT_SUMMARY in petri dishes until required. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cells were prepared for DESI MSI analysis by rinsing coverslips in 150 mM SP:SAMPLEPREP_SUMMARY ammonium acetate, pH 7.1 for 30 s before being allowed to dry in the air stream SP:SAMPLEPREP_SUMMARY of a biological safety cabinet for 15 min. Coverslips were then thoroughly dried SP:SAMPLEPREP_SUMMARY using a vacuum desiccator for 15 min before storage at -80 °C in petri dishes SP:SAMPLEPREP_SUMMARY until required. #CHROMATOGRAPHY CH:INSTRUMENT_NAME none CH:COLUMN_NAME none CH:CHROMATOGRAPHY_TYPE None (Direct infusion) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Xevo-G2-XS MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE Other MS:MS_COMMENTS Mass spectrometry imaging using 2D DESI source (Prosolia) was conducted at MS:MS_COMMENTS Waters Corporation (Wilmslow, UK). Dried cell cultures gown on coverslips were MS:MS_COMMENTS mounted on microscope glass slides using double sided tape. Slides were scanned MS:MS_COMMENTS using Epson perfection V600 photo scanner. Scanned images were imported and the MS:MS_COMMENTS area where cells were confluent was selected using the co-registered MS:MS_COMMENTS photographic image of the samples in High-Definition Imaging (HDI) 1.5. Imaging MS:MS_COMMENTS experiments were carried out on a DESI (Prosolia, USA) mounted on a Xevo-G2-XS MS:MS_COMMENTS quadrupole-time of flight (QTOF) mass spectrometer (Waters Corporation, MS:MS_COMMENTS Wilmslow, UK). The DESI spray was composed of a solvent mixture of 98:2% MeOH: MS:MS_COMMENTS water (v/v) delivered at a flow rate of 2 μl/min with nebulizing gas pressure MS:MS_COMMENTS of 5 bar. The sprayer geometric positions were set that the sprayer was 1.5 mm MS:MS_COMMENTS above sample surface and the distance between sprayer to capillary was 6mm. The MS:MS_COMMENTS source temperature was 100 °C. For both positive and negative ionization modes, MS:MS_COMMENTS the acquisition mass range was 50-1200 m/z. DESI MSI experiments were performed MS:MS_COMMENTS using the scan rate of 4 scan/ second in negative mode. The X and Y pixel sizes MS:MS_COMMENTS were set at 20 μm. Selected precursor ions in both positive and negative MS:MS_COMMENTS ionisation modes (including m/z 810.55 and m/z 773.53) were further analysed MS:MS_COMMENTS using MS/MS. The experiments were carried out on a DESI (Prosolia, place, USA) MS:MS_COMMENTS mounted on a Xevo-G2-XS Q-TOF mass spectrometer (Waters Corporation, Wilmslow, MS:MS_COMMENTS UK). Spray conditions were the same as DESI imaging. Spectra were visualised MS:MS_COMMENTS using MassLynx (Waters Corporation, Wilmslow, UK). Raw data from each biological MS:MS_COMMENTS condition were processed using HDI software version 1.5 (Waters Corporation, MS:MS_COMMENTS Wilmslow, UK) to provide ion images from a consolidated list of m/z common to MS:MS_COMMENTS the different datasets as well as the unique m/z. Ion images were normalised to MS:MS_COMMENTS total ion current (TIC). Regions of interest (ROIs) were drawn directly from the MS:MS_COMMENTS DESI images that produced a .csv file containing average TIC normalised MS:MS_COMMENTS intensities which was used for statistical analysis using MetaboAnalyst1 MS:MS_COMMENTS (https://www.metaboanalyst.ca/MetaboAnalyst/faces/home.xhtml) to compare between MS:MS_COMMENTS selected lipids that are presented in the two cell lines. Similarly, MSI MS:MS_COMMENTS analysis of Caco2/HT29-MTX co-culture was performed using the same criteria of MS:MS_COMMENTS DESI images with the same precursor ions mentioned above. For pixel MS:MS_COMMENTS classification of DESI imaging datasets, a Waters Corporation (WRC, Budapest, MS:MS_COMMENTS Hungary) prototype AMX MS imaging software was used in combination with HDI MS:MS_COMMENTS software. MS:ION_MODE NEGATIVE MS:MS_RESULTS_FILE ST002190_AN003584_Results.txt UNITS:Peak area Has m/z:Yes #END