Summary of Study ST002911

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001811. The data can be accessed directly via it's Project DOI: 10.21228/M8MQ7C This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST002911
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
Submit Date	2023-10-02
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	LC-MS
Release Date	2023-10-20
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001811
Project DOI:	doi: 10.21228/M8MQ7C
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.

Subject:

Subject ID:	SU003024
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

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Factor
SA316303	12_UT2	12-weeks infection
SA316304	12_UT4	12-weeks infection
SA316305	12_UT5	12-weeks infection
SA316306	12_UT1	12-weeks infection
SA316307	12_UT3	12-weeks infection
SA316308	1_UT6	4-weeks infection
SA316309	1_UT2	4-weeks infection
SA316310	1_UT3	4-weeks infection
SA316311	1_UT4	4-weeks infection
SA316312	1_UT5	4-weeks infection
SA316313	1_UT1	4-weeks infection
SA316314	1_UI6	control
SA316315	1_UI5	control
SA316316	1_UI1	control
SA316317	1_UI2	control
SA316318	1_UI3	control
SA316319	1_UI4	control

Showing results 1 to 17 of 17

Showing results 1 to 17 of 17

Collection:

Collection ID:	CO003017
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 ID:	TR003033
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.

Treatment ID:

TR003033

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.

Sample Preparation:

Sampleprep ID:	SP003030
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.

Sampleprep ID:

SP003030

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.

Combined analysis:

Analysis ID	AN004780	AN004781
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Agilent 1290 Infinity II	Agilent 1290 Infinity II
Column	Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um)	Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um)
MS Type	ESI	ESI
MS instrument type	QTOF	QTOF
MS instrument name	Agilent 6545 QTOF	Agilent 6545 QTOF
Ion Mode	POSITIVE	NEGATIVE
Units	AREA	AREA

Chromatography:

Chromatography ID:	CH003612
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3mm,2.7um)
Column Temperature:	50 °C
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
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
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004526
Analysis ID:	AN004780
Instrument Name:	Agilent 6545 QTOF
Instrument Type:	QTOF
MS Type:	ESI
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 121.0509 and HP-0921 (C18H18O6N3P3F24) at m/z 922.0098. 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.
Ion Mode:	POSITIVE

MS ID:	MS004527
Analysis ID:	AN004781
Instrument Name:	Agilent 6545 QTOF
Instrument Type:	QTOF
MS Type:	ESI
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.
Ion Mode:	NEGATIVE