Summary of Study ST002438

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 PR001570. The data can be accessed directly via it's Project DOI: 10.21228/M8RM66 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	ST002438
Study Title	Ozone alters glycosphingolipid metabolism and exacerbates characteristics of asthma in mice
Study Summary	Asthma is a common chronic respiratory disease exacerbated by multiple environmental factors, including exposure to air pollutants such as ozone. Acute ozone exposure has previously been implicated in airway inflammation, airway hyperreactivity, and other characteristics of asthma. Altered sphingolipid metabolism following ozone exposure may contribute to the molecular mechanisms underlying these previously reported effects. This study aimed to identify changes in metabolomic profiles and characteristics of asthma in allergen-sensitized mice following ozone exposure to provide insights regarding mechanisms of ozone-induced exacerbations in asthma. Adult male and female BALB/c mice were sensitized intranasally to house dust mite allergen (HDM) on days 1, 3, and 5 followed by HDM challenge on days 12-14. Mice were subsequently exposed to ozone following each HDM challenge for 6 hr/day. Bronchoalveolar lavage, plasma, whole lung lobes, and microdissected lung airways were collected from 8 female and 8 male mice for metabolomics analysis. 6 female and 6 male mice underwent methacholine challenge using a forced oscillation technique to assess pulmonary function. HDM-sensitized male mice exposed to ozone displayed synergistically increased airway hyperreactivity as well as increased airway inflammation and eosinophilia relative to control mice. Effects in male mice were significantly more severe than the effects observed in females. Both HDM-sensitized male and female mice exposed to ozone displayed significant decreases in multiple classes of sphingolipids in microdissected airways. However, glycosphingolipids were significantly increased in females and to a lesser extent in males. These results potentially implicate glycosphingolipids in protecting against severe outcomes of ozone exposure that coincide with exacerbation of allergic asthma.
Institute	University of California, Davis
Last Name	Stevens
First Name	Nathanial
Address	451 Health Sciences Drive
Email	ncstevens@ucdavis.edu
Phone	8282844315
Submit Date	2022-08-25
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2023-01-25
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001570
Project DOI:	doi: 10.21228/M8RM66
Project Title:	Ozone alters glycosphingolipid metabolism and exacerbates characteristics of asthma in mice
Project Summary:	Asthma is a common chronic respiratory disease exacerbated by multiple environmental factors, including exposure to air pollutants such as ozone. Acute ozone exposure has previously been implicated in airway inflammation, airway hyperreactivity, and other characteristics of asthma. Altered sphingolipid metabolism following ozone exposure may contribute to the molecular mechanisms underlying these previously reported effects. This study aimed to identify changes in metabolomic profiles and characteristics of asthma in allergen-sensitized mice following ozone exposure to provide insights regarding mechanisms of ozone-induced exacerbations in asthma. Adult male and female BALB/c mice were sensitized intranasally to house dust mite allergen (HDM) on days 1, 3, and 5 followed by HDM challenge on days 12-14. Mice were subsequently exposed to ozone following each HDM challenge for 6 hr/day. Bronchoalveolar lavage, plasma, whole lung lobes, and microdissected lung airways were collected from 8 female and 8 male mice for metabolomics analysis. 6 female and 6 male mice underwent methacholine challenge using a forced oscillation technique to assess pulmonary function. HDM-sensitized male mice exposed to ozone displayed synergistically increased airway hyperreactivity as well as increased airway inflammation and eosinophilia relative to control mice. Effects in male mice were significantly more severe than the effects observed in females. Both HDM-sensitized male and female mice exposed to ozone displayed significant decreases in multiple classes of sphingolipids in microdissected airways. However, glycosphingolipids were significantly increased in females and to a lesser extent in males. These results potentially implicate glycosphingolipids in protecting against severe outcomes of ozone exposure that coincide with exacerbation of allergic asthma.
Institute:	University of California Davis
Last Name:	Stevens
First Name:	Nathanial
Address:	451 Health Sciences Drive, Davis, CA, 95616, USA
Email:	ncstevens@ucdavis.edu
Phone:	8282844315
Funding Source:	R21 ES030276, T32 ES007059, T32 HL007013, NIH U19 AG023122

Subject:

Subject ID:	SU002527
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	BALB/c
Age Or Age Range:	8-10 wks.
Gender:	Male and female
Animal Animal Supplier:	Envigo
Animal Light Cycle:	12/12 light/dark

Factors:

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

mb_sample_id	local_sample_id	Factor
SA243615	mx626289_OzAW_BR3	BR
SA243616	mx626289_OzAW_BR1	BR
SA243617	mx626289_OzAW_BR4	BR
SA243618	mx626289_OzAW_BR2	BR
SA243619	mx626289_OzAW_BR7	BR
SA243620	mx626289_OzAW_BR6	BR
SA243621	mx626289_OzAW_BR5	BR
SA243622	mx626289_OzAW_75	HFA_F
SA243623	mx626289_OzAW_74	HFA_F
SA243624	mx626289_OzAW_76	HFA_F
SA243625	mx626289_OzAW_80	HFA_F
SA243626	mx626289_OzAW_73	HFA_F
SA243627	mx626289_OzAW_79	HFA_F
SA243628	mx626289_OzAW_78	HFA_F
SA243629	mx626289_OzAW_77	HFA_F
SA243630	mx626289_OzAW_65	HFA_M
SA243631	mx626289_OzAW_70	HFA_M
SA243632	mx626289_OzAW_71	HFA_M
SA243633	mx626289_OzAW_66	HFA_M
SA243634	mx626289_OzAW_67	HFA_M
SA243635	mx626289_OzAW_69	HFA_M
SA243636	mx626289_OzAW_68	HFA_M
SA243637	mx626289_OzAW_72	HFA_M
SA243638	mx626289_OzAW_89	HO3_F
SA243639	mx626289_OzAW_94	HO3_F
SA243640	mx626289_OzAW_95	HO3_F
SA243641	mx626289_OzAW_96	HO3_F
SA243642	mx626289_OzAW_93	HO3_F
SA243643	mx626289_OzAW_92	HO3_F
SA243644	mx626289_OzAW_90	HO3_F
SA243645	mx626289_OzAW_91	HO3_F
SA243646	mx626289_OzAW_83	HO3_M
SA243647	mx626289_OzAW_82	HO3_M
SA243648	mx626289_OzAW_86	HO3_M
SA243649	mx626289_OzAW_88	HO3_M
SA243650	mx626289_OzAW_87	HO3_M
SA243651	mx626289_OzAW_81	HO3_M
SA243652	mx626289_OzAW_85	HO3_M
SA243653	mx626289_OzAW_84	HO3_M
SA243654	mx626289_OzAW_MB06	MB
SA243655	mx626289_OzAW_MB07	MB
SA243656	mx626289_OzAW_MB05	MB
SA243657	mx626289_OzAW_MB04	MB
SA243658	mx626289_OzAW_MB01	MB
SA243659	mx626289_OzAW_MB02	MB
SA243660	mx626289_OzAW_MB03	MB
SA243661	mx626289_OzAW_P1	P
SA243662	mx626289_OzAW_P3	P
SA243663	mx626289_OzAW_P2	P
SA243664	mx626289_OzAW_P5	P
SA243665	mx626289_OzAW_P7	P
SA243666	mx626289_OzAW_P4	P
SA243667	mx626289_OzAW_P6	P
SA243668	mx626289_OzAW_16	SFA_F
SA243669	mx626289_OzAW_11	SFA_F
SA243670	mx626289_OzAW_10	SFA_F
SA243671	mx626289_OzAW_15	SFA_F
SA243672	mx626289_OzAW_14	SFA_F
SA243673	mx626289_OzAW_12	SFA_F
SA243674	mx626289_OzAW_9	SFA_F
SA243675	mx626289_OzAW_13	SFA_F
SA243676	mx626289_OzAW_1	SFA_M
SA243677	mx626289_OzAW_6	SFA_M
SA243678	mx626289_OzAW_5	SFA_M
SA243679	mx626289_OzAW_4	SFA_M
SA243680	mx626289_OzAW_3	SFA_M
SA243681	mx626289_OzAW_7	SFA_M
SA243682	mx626289_OzAW_8	SFA_M
SA243683	mx626289_OzAW_2	SFA_M
SA243684	mx626289_OzAW_26	SO3_F
SA243685	mx626289_OzAW_29	SO3_F
SA243686	mx626289_OzAW_30	SO3_F
SA243687	mx626289_OzAW_32	SO3_F
SA243688	mx626289_OzAW_28	SO3_F
SA243689	mx626289_OzAW_31	SO3_F
SA243690	mx626289_OzAW_25	SO3_F
SA243691	mx626289_OzAW_27	SO3_F
SA243692	mx626289_OzAW_18	SO3_M
SA243693	mx626289_OzAW_19	SO3_M
SA243694	mx626289_OzAW_20	SO3_M
SA243695	mx626289_OzAW_22	SO3_M
SA243696	mx626289_OzAW_24	SO3_M
SA243697	mx626289_OzAW_21	SO3_M
SA243698	mx626289_OzAW_17	SO3_M
SA243699	mx626289_OzAW_23	SO3_M

Showing results 1 to 85 of 85

Showing results 1 to 85 of 85

Collection:

Collection ID:	CO002520
Collection Summary:	Right lung lobes were microdissected following a previous protocol (Plopper et al. 1991; Stevens et al. 2021). Briefly, 3 right lobes from each mouse collected during necropsy were blunt dissected on ice by removal of the surrounding lung parenchyma from the airways. The dissected lung airways were promptly transferred and stored at -80 degrees C until preparation for metabolomics analysis.
Sample Type:	Lung airways

Treatment:

Treatment ID:	TR002539
Treatment Summary:	Mice underwent intranasal instillation with either house-dust mite dissolved in PBS or vehicle on days 1,3, and 5. Mice were challenged with HDM or vehicle on days 12-14 and subsequently exposed to either filtered air or ozone (0.5ppm, 6hr./day) after each challenge.

Sample Preparation:

Sampleprep ID:	SP002533
Sampleprep Summary:	Extraction of Mammalian Tissue Samples: Liver 1. References: Fiehn O, Kind T (2006) Metabolite profiling in blood plasma. In: Metabolomics: Methods and Protocols. Weckwerth W (ed.), Humana Press, Totowa NJ (in press) 2.Starting material: Liver sample: weigh 4mg per sample into 2mL Eppendorf tubes. 3. Equipment: Centrifuge (Eppendorf 5415 D) Calibrated pipettes 1-200μl and 100-1000μl Eppendorf tubes 2mL, clear (Cat. No. 022363204) Centrifuge tubes 50mL, polypropylene Eppendorff Tabletop Centrifuge (Proteomics core Lab.) ThermoElectron Neslab RTE 740 cooling bath at –20°C MiniVortexer (VWR) Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments) Speed vacuum concentration system (Labconco Centrivap cold trap) Turex mini homogenizer 4. Chemicals Acetonitrile, LCMS grade (JT Baker; Cat. No.9829-02) Isopropanol, HPLC grade (JT Baker; Cat. No. 9095-02) Methanol Acetone Crushed ice 18 MΩ pure water (Millipore) Nitrogen line with pipette tip pH paper 5-10 (EMD Chem. Inc.) 5. Procedure Preparation of extraction mix and material before experiment: Switch on bath to pre-cool at –20°C (±2°C validity temperature range) Check pH of acetonitrile and isopropanol (pH7) using wetted pH paper Make the extraction solution by mixing acetonitrile, isopropanol and water in proportions 3 : 3 : 2 De-gas the extraction solution for 5 min with nitrogen. Make sure that the nitrogen line was flushed out of air before using it for degassing the extraction solvent solution Sample Preparation Weigh 4mg tissue sample in to a 2mL Eppendorf tube. Add 1mL extraction solvent to the tissue sample and homogenize for 45 seconds ensuring that sample resembles a powder. In between samples, clean the homogenizer in solutions of methanol, acetone, water, and the extraction solvent in the order listed. Vortex samples for 10 seconds, then 5 minutes on 4°C shaker. Centrifuge the samples for 2 minutes at 14,000 rcf. Aliquot 500µL supernatant for analysis, and 500µL for a backup. Store backup aliquots in the -20°C freezer. Evaporate one 500µl analysis aliquot in the Labconco Centrivap cold trap concentrator to complete dryness (typically overnight). The dried aliquot is then re-suspended with 500μl 50% acetonitrile (degassed as given) Centrifuge for 2 minutes at 14,000 rcf using the centrifuge Eppendorf 5415. Remove supernatant to a new Eppendorf tube. Evaporate the supernatant to dryness in the the Labconco Centrivap cold trap concentrator. Submit to derivatization. The residue should contain membrane lipids because these are supposedly not soluble enough to be found in the 50% acetonitrile solution. Therefore, this ‘membrane residue’ is now taken for membrane lipidomic fingerprinting using the nanomate LTQ ion trap mass spectrometer. Likely, a good solvent to redissolve the membrane lipids is e.g. 75% isopropanol (degassed as given above). If the ‘analysis’ aliquot is to be used for semi lipophilic compounds such as tyrosine pathway intermediates (incl. dopamine, serotonine etc, i.e. polar aromatic compounds), then these are supposedly to be found together with the ‘GCTOF’ aliquot. We can assume that this mixture is still too complex for Agilent chipLCMS. Therefore, in order to develop and validate target analysis for such aromatic compounds, we should use some sort of Solid Phase purification. We re-suspend the dried ‘GCTOF’ aliquot in 300 l water (degassed as before) to take out sugars, aliphatic amino acids, hydroxyl acids and similar logP compounds. The residue should contain our target aromatics .We could also try to adjust pH by using low concentration acetate or phosphate buffer. The residue could then be taken up in 50% acetonitrile and used for GCTOF and Agilent chipMS experiments. The other aliquot should be checked how much of our target compounds would actually be found in the ‘sugar’ fraction. 6. Problems To prevent contamination disposable material is used. Control pH from extraction mix. 7. Quality assurance For each sequence of sample extractions, perform one blank negative control extraction by applying the total procedure (i.e. all materials and plastic ware) without biological sample. 8. Disposal of waste Collect all chemicals in appropriate bottles and follow the disposal rules.

Sampleprep ID:

SP002533

Sampleprep Summary:

Extraction of Mammalian Tissue Samples: Liver 1. References: Fiehn O, Kind T (2006) Metabolite profiling in blood plasma. In: Metabolomics: Methods and Protocols. Weckwerth W (ed.), Humana Press, Totowa NJ (in press) 2.Starting material: Liver sample: weigh 4mg per sample into 2mL Eppendorf tubes. 3. Equipment: Centrifuge (Eppendorf 5415 D) Calibrated pipettes 1-200μl and 100-1000μl Eppendorf tubes 2mL, clear (Cat. No. 022363204) Centrifuge tubes 50mL, polypropylene Eppendorff Tabletop Centrifuge (Proteomics core Lab.) ThermoElectron Neslab RTE 740 cooling bath at –20°C MiniVortexer (VWR) Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments) Speed vacuum concentration system (Labconco Centrivap cold trap) Turex mini homogenizer 4. Chemicals Acetonitrile, LCMS grade (JT Baker; Cat. No.9829-02) Isopropanol, HPLC grade (JT Baker; Cat. No. 9095-02) Methanol Acetone Crushed ice 18 MΩ pure water (Millipore) Nitrogen line with pipette tip pH paper 5-10 (EMD Chem. Inc.) 5. Procedure Preparation of extraction mix and material before experiment: Switch on bath to pre-cool at –20°C (±2°C validity temperature range) Check pH of acetonitrile and isopropanol (pH7) using wetted pH paper Make the extraction solution by mixing acetonitrile, isopropanol and water in proportions 3 : 3 : 2 De-gas the extraction solution for 5 min with nitrogen. Make sure that the nitrogen line was flushed out of air before using it for degassing the extraction solvent solution Sample Preparation Weigh 4mg tissue sample in to a 2mL Eppendorf tube. Add 1mL extraction solvent to the tissue sample and homogenize for 45 seconds ensuring that sample resembles a powder. In between samples, clean the homogenizer in solutions of methanol, acetone, water, and the extraction solvent in the order listed. Vortex samples for 10 seconds, then 5 minutes on 4°C shaker. Centrifuge the samples for 2 minutes at 14,000 rcf. Aliquot 500µL supernatant for analysis, and 500µL for a backup. Store backup aliquots in the -20°C freezer. Evaporate one 500µl analysis aliquot in the Labconco Centrivap cold trap concentrator to complete dryness (typically overnight). The dried aliquot is then re-suspended with 500μl 50% acetonitrile (degassed as given) Centrifuge for 2 minutes at 14,000 rcf using the centrifuge Eppendorf 5415. Remove supernatant to a new Eppendorf tube. Evaporate the supernatant to dryness in the the Labconco Centrivap cold trap concentrator. Submit to derivatization. The residue should contain membrane lipids because these are supposedly not soluble enough to be found in the 50% acetonitrile solution. Therefore, this ‘membrane residue’ is now taken for membrane lipidomic fingerprinting using the nanomate LTQ ion trap mass spectrometer. Likely, a good solvent to redissolve the membrane lipids is e.g. 75% isopropanol (degassed as given above). If the ‘analysis’ aliquot is to be used for semi lipophilic compounds such as tyrosine pathway intermediates (incl. dopamine, serotonine etc, i.e. polar aromatic compounds), then these are supposedly to be found together with the ‘GCTOF’ aliquot. We can assume that this mixture is still too complex for Agilent chipLCMS. Therefore, in order to develop and validate target analysis for such aromatic compounds, we should use some sort of Solid Phase purification. We re-suspend the dried ‘GCTOF’ aliquot in 300 l water (degassed as before) to take out sugars, aliphatic amino acids, hydroxyl acids and similar logP compounds. The residue should contain our target aromatics .We could also try to adjust pH by using low concentration acetate or phosphate buffer. The residue could then be taken up in 50% acetonitrile and used for GCTOF and Agilent chipMS experiments. The other aliquot should be checked how much of our target compounds would actually be found in the ‘sugar’ fraction. 6. Problems To prevent contamination disposable material is used. Control pH from extraction mix. 7. Quality assurance For each sequence of sample extractions, perform one blank negative control extraction by applying the total procedure (i.e. all materials and plastic ware) without biological sample. 8. Disposal of waste Collect all chemicals in appropriate bottles and follow the disposal rules.

Combined analysis:

Analysis ID	AN003972	AN003973	AN003974	AN003975
Analysis type	MS	MS	MS	MS
Chromatography type	Reversed phase	Reversed phase	HILIC	HILIC
Chromatography system	Thermo Vanquish	Thermo Vanquish	Agilent 6490	Agilent 6490
Column	Waters Acquity CSH C18 (100 x 2.1mm, 1.7um)	Waters Acquity CSH C18 (100 x 2.1mm, 1.7um)	Agilent HP5-MS (30m x 0.25mm, 0.25 um)	Agilent HP5-MS (30m x 0.25mm, 0.25 um)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	Orbitrap	Orbitrap	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap
Ion Mode	POSITIVE	NEGATIVE	POSITIVE	NEGATIVE
Units	Relative abundance	Relative abundance	Relative abundance	Relative abundance

Chromatography:

Chromatography ID:	CH002937
Instrument Name:	Thermo Vanquish
Column Name:	Waters Acquity CSH C18 (100 x 2.1mm, 1.7um)
Chromatography Type:	Reversed phase

Chromatography ID:	CH002938
Instrument Name:	Agilent 6490
Column Name:	Agilent HP5-MS (30m x 0.25mm, 0.25 um)
Chromatography Type:	HILIC

MS:

MS ID:	MS003706
Analysis ID:	AN003972
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The ion source conditions were set as follows: spray voltage, -3.0 kV; sheath gas flow rate, 60 arbitrary units; aux gas flow rate, 25 arbitrary units; sweep gas flow rate, 2 arbitrary units; capillary temp, 300 °C; S-lens RF level, 50; Aux gas heater temp, 370 °C. The following acquisition parameters were used for MS1 analysis: resolution, 60000, AGC target, 1e6; Maximum IT, 100 ms; scan range 60-900 m/z; spectrum data type, centroid. Data dependent MS/MS parameters: resolution, 15000; AGC target, 1e5; maximum IT, 50 ms; loop count, 4; TopN, 4; isolation window, 1.0 m/z; fixed first mass, 70.0 m/z; (N)CE/ stepped nce, 20, 30, 40; spectrum data type, centroid; minimum AGC target, 8e3; intensity threshold, 1.6e5; exclude isotopes, on; dynamic exclusion, 3.0 s. To increase the total number of MS/MS spectra, five runs with iterative MS/MS exclusions were performed using the R package “IE-Omics”18 for both positive and negative electrospray conditions.
Ion Mode:	POSITIVE

MS ID:	MS003707
Analysis ID:	AN003973
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The ion source conditions were set as follows: spray voltage, -3.0 kV; sheath gas flow rate, 60 arbitrary units; aux gas flow rate, 25 arbitrary units; sweep gas flow rate, 2 arbitrary units; capillary temp, 300 °C; S-lens RF level, 50; Aux gas heater temp, 370 °C. The following acquisition parameters were used for MS1 analysis: resolution, 60000, AGC target, 1e6; Maximum IT, 100 ms; scan range 60-900 m/z; spectrum data type, centroid. Data dependent MS/MS parameters: resolution, 15000; AGC target, 1e5; maximum IT, 50 ms; loop count, 4; TopN, 4; isolation window, 1.0 m/z; fixed first mass, 70.0 m/z; (N)CE/ stepped nce, 20, 30, 40; spectrum data type, centroid; minimum AGC target, 8e3; intensity threshold, 1.6e5; exclude isotopes, on; dynamic exclusion, 3.0 s. To increase the total number of MS/MS spectra, five runs with iterative MS/MS exclusions were performed using the R package “IE-Omics”18 for both positive and negative electrospray conditions.
Ion Mode:	NEGATIVE

MS ID:	MS003708
Analysis ID:	AN003974
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The ion source conditions were set as follows: spray voltage, -3.0 kV; sheath gas flow rate, 60 arbitrary units; aux gas flow rate, 25 arbitrary units; sweep gas flow rate, 2 arbitrary units; capillary temp, 300 °C; S-lens RF level, 50; Aux gas heater temp, 370 °C. The following acquisition parameters were used for MS1 analysis: resolution, 60000, AGC target, 1e6; Maximum IT, 100 ms; scan range 60-900 m/z; spectrum data type, centroid. Data dependent MS/MS parameters: resolution, 15000; AGC target, 1e5; maximum IT, 50 ms; loop count, 4; TopN, 4; isolation window, 1.0 m/z; fixed first mass, 70.0 m/z; (N)CE/ stepped nce, 20, 30, 40; spectrum data type, centroid; minimum AGC target, 8e3; intensity threshold, 1.6e5; exclude isotopes, on; dynamic exclusion, 3.0 s. To increase the total number of MS/MS spectra, five runs with iterative MS/MS exclusions were performed using the R package “IE-Omics”18 for both positive and negative electrospray conditions.
Ion Mode:	POSITIVE

MS ID:	MS003709
Analysis ID:	AN003975
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The ion source conditions were set as follows: spray voltage, -3.0 kV; sheath gas flow rate, 60 arbitrary units; aux gas flow rate, 25 arbitrary units; sweep gas flow rate, 2 arbitrary units; capillary temp, 300 °C; S-lens RF level, 50; Aux gas heater temp, 370 °C. The following acquisition parameters were used for MS1 analysis: resolution, 60000, AGC target, 1e6; Maximum IT, 100 ms; scan range 60-900 m/z; spectrum data type, centroid. Data dependent MS/MS parameters: resolution, 15000; AGC target, 1e5; maximum IT, 50 ms; loop count, 4; TopN, 4; isolation window, 1.0 m/z; fixed first mass, 70.0 m/z; (N)CE/ stepped nce, 20, 30, 40; spectrum data type, centroid; minimum AGC target, 8e3; intensity threshold, 1.6e5; exclude isotopes, on; dynamic exclusion, 3.0 s. To increase the total number of MS/MS spectra, five runs with iterative MS/MS exclusions were performed using the R package “IE-Omics”18 for both positive and negative electrospray conditions.
Ion Mode:	NEGATIVE