Summary of Study ST003769
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 PR002351. The data can be accessed directly via it's Project DOI: 10.21228/M8R53K 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 | ST003769 |
| Study Title | Characterization of brain‐derived extracellular vesicle lipids in Alzheimer's disease |
| Study Summary | Lipid dyshomeostasis is associated with the most common form of dementia, Alzheimer's disease (AD). Substantial progress has been made in identifying positron emission tomography and cerebrospinal fluid biomarkers for AD, but they have limited use as front‐line diagnostic tools. Extracellular vesicles (EVs) are released by all cells and contain a subset of their parental cell composition, including lipids. EVs are released from the brain into the periphery, providing a potential source of tissue and disease specific lipid biomarkers. However, the EV lipidome of the central nervous system is currently unknown and the potential of brain‐derived EVs (BDEVs) to inform on lipid dyshomeostasis in AD remains unclear. The aim of this study was to reveal the lipid composition of BDEVs in human frontal cortex, and to determine whether BDEVs have an altered lipid profile in AD. Using semi‐quantitative mass spectrometry, we describe the BDEV lipidome, covering four lipid categories, 17 lipid classes and 692 lipid molecules. BDEVs were enriched in glycerophosphoserine (PS) lipids, a characteristic of small EVs. Here we further report that BDEVs are enriched in ether‐containing PS lipids, a finding that further establishes ether lipids as a feature of EVs. BDEVs in the AD frontal cortex offered improved detection of dysregulated lipids in AD over global lipid profiling of this brain region. AD BDEVs had significantly altered glycerophospholipid and sphingolipid levels, specifically increased plasmalogen glycerophosphoethanolamine and decreased polyunsaturated fatty acyl containing lipids, and altered amide‐linked acyl chain content in sphingomyelin and ceramide lipids relative to CTL. The most prominent alteration was a two‐fold decrease in lipid species containing anti‐inflammatory/pro‐resolving docosahexaenoic acid. The in‐depth lipidome analysis provided in this study highlights the advantage of EVs over more complex tissues for improved detection of dysregulated lipids that may serve as potential biomarkers in the periphery. |
| Institute | University of Melbourne |
| Department | The Florey Institute of Neuroscience and Mental Health |
| Last Name | Su |
| First Name | Huaqi |
| Address | 30 Royal Parade, Melbourne, VIC, 3150, Australia |
| huaqi.su@unimelb.edu.au | |
| Phone | +61416373787 |
| Submit Date | 2025-02-25 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2025-03-25 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002351 |
| Project DOI: | doi: 10.21228/M8R53K |
| Project Title: | Characterization of brain‐derived extracellular vesicle lipids in Alzheimer's disease |
| Project Type: | MS lipidomic quantitative analysis |
| Project Summary: | Lipid dyshomeostasis is associated with the most common form of dementia, Alzheimer's disease (AD). Substantial progress has been made in identifying positron emission tomography and cerebrospinal fluid biomarkers for AD, but they have limited use as front‐line diagnostic tools. Extracellular vesicles (EVs) are released by all cells and contain a subset of their parental cell composition, including lipids. EVs are released from the brain into the periphery, providing a potential source of tissue and disease specific lipid biomarkers. However, the EV lipidome of the central nervous system is currently unknown and the potential of brain‐derived EVs (BDEVs) to inform on lipid dyshomeostasis in AD remains unclear. The aim of this study was to reveal the lipid composition of BDEVs in human frontal cortex, and to determine whether BDEVs have an altered lipid profile in AD. Using semi‐quantitative mass spectrometry, we describe the BDEV lipidome, covering four lipid categories, 17 lipid classes and 692 lipid molecules. BDEVs were enriched in glycerophosphoserine (PS) lipids, a characteristic of small EVs. Here we further report that BDEVs are enriched in ether‐containing PS lipids, a finding that further establishes ether lipids as a feature of EVs. BDEVs in the AD frontal cortex offered improved detection of dysregulated lipids in AD over global lipid profiling of this brain region. AD BDEVs had significantly altered glycerophospholipid and sphingolipid levels, specifically increased plasmalogen glycerophosphoethanolamine and decreased polyunsaturated fatty acyl containing lipids, and altered amide‐linked acyl chain content in sphingomyelin and ceramide lipids relative to CTL. The most prominent alteration was a two‐fold decrease in lipid species containing anti‐inflammatory/pro‐resolving docosahexaenoic acid. The in‐depth lipidome analysis provided in this study highlights the advantage of EVs over more complex tissues for improved detection of dysregulated lipids that may serve as potential biomarkers in the periphery. |
| Institute: | The University of Melbourne |
| Department: | The Florey Institute of Neuroscience and Mental Health |
| Last Name: | Su |
| First Name: | Huaqi |
| Address: | 30 Royal Parade, Melbourne, VIC, 3150, Australia |
| Email: | huaqi.su@unimelb.edu.au |
| Phone: | +61416373787 |
Subject:
| Subject ID: | SU003902 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Male |
Factors:
Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Diagnosis |
|---|---|---|---|
| SA409610 | 06379AD_F2 | BDEV | AD |
| SA409611 | V15016AD_F2 | BDEV | AD |
| SA409612 | 09214AD_F2 | BDEV | AD |
| SA409613 | 09090AD_F2 | BDEV | AD |
| SA409614 | 09006AD_F2 | BDEV | AD |
| SA409615 | 08329AD_F2 | BDEV | AD |
| SA409616 | 08312AD_F2 | BDEV | AD |
| SA409617 | 08007AD_F2 | BDEV | AD |
| SA409618 | V14035CTL_F2 | BDEV | CTL |
| SA409619 | V13052CTL_F2 | BDEV | CTL |
| SA409620 | V11052CTL_F2 | BDEV | CTL |
| SA409621 | 07635CTL_F2 | BDEV | CTL |
| SA409622 | 07284CTL_F2 | BDEV | CTL |
| SA409623 | 07022CTL_F2 | BDEV | CTL |
| SA409624 | 06972CTL_F2 | BDEV | CTL |
| SA409625 | 051071CTL_F2 | BDEV | CTL |
| SA409626 | 06379AD_T | Brain | AD |
| SA409627 | V15016AD_T | Brain | AD |
| SA409628 | 09214AD_T | Brain | AD |
| SA409629 | 09090AD_T | Brain | AD |
| SA409630 | 09006AD_T | Brain | AD |
| SA409631 | 08329AD_T | Brain | AD |
| SA409632 | 08312AD_T | Brain | AD |
| SA409633 | 08007AD_T | Brain | AD |
| SA409634 | V14035CTL_T | Brain | CTL |
| SA409635 | V13052CTL_T | Brain | CTL |
| SA409636 | V11052CTL_T | Brain | CTL |
| SA409637 | 07635CTL_T | Brain | CTL |
| SA409638 | 07284CTL_T | Brain | CTL |
| SA409639 | 07022CTL_T | Brain | CTL |
| SA409640 | 06972CTL_T | Brain | CTL |
| SA409641 | 051071CTL_T | Brain | CTL |
| Showing results 1 to 32 of 32 |
Collection:
| Collection ID: | CO003895 |
| Collection Summary: | Fresh frozen human post-mortem frontal cortex tissues of n = 8 AD male subjects (mean age 74.5 ± SD 7.0 years) and n = 8 gender and age-matched CTL subjects (mean age 73.5 ± SD 5.9 years) with no evidence of dementia, stored at −80◦C, were obtained from the Victoria Brain Bank. The average post-mortem delay before tissue collection was 23.3 ± 17.4 h for AD and 42 ± 16.3 h for CTL. Frozen frontal cortex tissues (approximately 2 g) were sliced lengthways on ice to generate 1–2 cm long, 2–3 mm wide tissue sections. Approximately 30 mg tissue pieces from each sample (“Brain Total”) were collected, weighed and placed in 19x volume of tissue weight of Dulbecco’s phosphate buffered saline (DPBS, Thermo Fisher Scientific) solution containing 1x PhosSTOP phosphatase inhibitor (Sigma Aldrich) / cOmplete protease inhibitor (including EDTA, Sigma Aldrich) for immunoblot analysis. The remaining cut tissue sections were weighed and incubated with 50 U/ml collagenase type 3 (#CLS-3, CAT#LS004182, Worthington) digestion buffer (at ratio of 8μl / mg tissue) in a shaking water bath (25◦C, a total of 20 min). During incubation, tissue slices were inverted twice at the 10-min time point, gently pipetted up and down twice at the 15-min time point and then allowed incubation for a further 5 min, followed by the addition of ice-cold 10x inhibition buffer, which was made of 10x phosphatase inhibitor and 10x protease inhibitor in DPBS. The final concentration of inhibition buffer in solution was 1x. The dissociated tissue in solution was subjected to a series of centrifugations, including a 300 × g, 4◦C for 5 min, a 2000 × g, 4◦C for 10 min and a 10,000 × g, 4◦C for 30 min. Representative 300 × g pellets were collected (‘Brain+C’ for collagenase treatment) and either placed in 19× volume of tissue weight of DPBS with 1× phosphatase inhibitor / protease inhibitor solution for protein quantification and immunoblot analysis or combined with 19× volume of tissue weight of ice-cold 60% methanol (LCMS grade, EMD Millipore Corporation) containing 0.01% (w/v) butylated hydroxytoluene (BHT, Sigma Aldrich) for lipid extraction. The 10,000 × g supernatant was loaded on top of the triple sucrose density gradient (0.6 M, 1.3M, 2.5 M) as indicated in the method (Vella et al., 2017) in ultra-clear SW40Ti tubes (Beckman Coulter). The sucrose gradients were centrifuged at 200,000 x g avg at 4◦C for 173 min using a SW40Ti rotor (Beckman Coulter). After the spin, the three fractions (F1, F2 and F3, 1.2ml each) were sequentially collected and refractive index wasmeasured. Each fraction was subjected to a wash spin in ice-cold DPBS at 128,000 × g avg, at 4◦C for 80 min using a F37L-8 × 100 rotor (Thermo Fisher Scientific). The pelleted EVs were resuspended in 150 μl ice-cold DPBS with 1x phosphatase inhibitor / protease inhibitor solution. |
| Collection Protocol Filename: | Su_et_al_2021.pdf |
| Sample Type: | brain frontal cortex, brain derived extracellular vesicles |
Treatment:
| Treatment ID: | TR003911 |
| Treatment Summary: | There is no treatment in this study. |
Sample Preparation:
| Sampleprep ID: | SP003908 |
| Sampleprep Summary: | Fresh frozen human post-mortem frontal cortex tissues of n = 8 AD male subjects (mean age 74.5 ± SD 7.0 years) and n = 8 gender and age-matched CTL subjects (mean age 73.5 ± SD 5.9 years) with no evidence of dementia, stored at −80◦C, were obtained from the Victoria Brain Bank. The average post-mortem delay before tissue collection was 23.3 ± 17.4 h for AD and 42 ± 16.3 h for CTL. Frozen frontal cortex tissues (approximately 2 g) were sliced lengthways on ice to generate 1–2 cm long, 2–3 mm wide tissue sections. Approximately 30 mg tissue pieces from each sample (“Brain Total”) were collected, weighed and placed in 19x volume of tissue weight of Dulbecco’s phosphate buffered saline (DPBS, Thermo Fisher Scientific) solution containing 1x PhosSTOP phosphatase inhibitor (Sigma Aldrich) / cOmplete protease inhibitor (including EDTA, Sigma Aldrich) for immunoblot analysis. The remaining cut tissue sections were weighed and incubated with 50 U/ml collagenase type 3 (#CLS-3, CAT#LS004182, Worthington) digestion buffer (at ratio of 8μl / mg tissue) in a shaking water bath (25◦C, a total of 20 min). During incubation, tissue slices were inverted twice at the 10-min time point, gently pipetted up and down twice at the 15-min time point and then allowed incubation for a further 5 min, followed by the addition of ice-cold 10x inhibition buffer, which was made of 10x phosphatase inhibitor and 10x protease inhibitor in DPBS. The final concentration of inhibition buffer in solution was 1x. The dissociated tissue in solution was subjected to a series of centrifugations, including a 300 × g, 4◦C for 5 min, a 2000 × g, 4◦C for 10 min and a 10,000 × g, 4◦C for 30 min. Representative 300 × g pellets were collected (‘Brain+C’ for collagenase treatment) and either placed in 19× volume of tissue weight of DPBS with 1× phosphatase inhibitor / protease inhibitor solution for protein quantification and immunoblot analysis or combined with 19× volume of tissue weight of ice-cold 60% methanol (LCMS grade, EMD Millipore Corporation) containing 0.01% (w/v) butylated hydroxytoluene (BHT, Sigma Aldrich) for lipid extraction. The 10,000 × g supernatant was loaded on top of the triple sucrose density gradient (0.6 M, 1.3M, 2.5 M) as indicated in the method (Vella et al., 2017) in ultra-clear SW40Ti tubes (Beckman Coulter). The sucrose gradients were centrifuged at 200,000 x g avg at 4◦C for 173 min using a SW40Ti rotor (Beckman Coulter). After the spin, the three fractions (F1, F2 and F3, 1.2ml each) were sequentially collected and refractive index wasmeasured. Each fraction was subjected to a wash spin in ice-cold DPBS at 128,000 × g avg, at 4◦C for 80 min using a F37L-8 × 100 rotor (Thermo Fisher Scientific). The pelleted EVs were resuspended in 150 μl ice-cold DPBS with 1x phosphatase inhibitor / protease inhibitor solution. |
| Sampleprep Protocol Filename: | Su_et_al_2021.pdf |
| Extraction Method: | The “total brain with collagenase” tissue pellets in ice-cold 60%methanol containing 0.01% (w/v) BHTwere homogenised using a cell disrupter as described above. 100 μl of the homogenates were combined with 100 μl of 60%methanol containing 0.01% (w/v) BHT. 80 μl of the F2BDEVsuspensionswere combinedwith 20 μl of ice-coldmethanolwith0.1%(w/v)BHTand100μl of ice-cold methanol to make a final volume of 200 μl 60%methanol containing 0.01% (w/v) BHT. All samples were sonicated in an ice-cold water bath sonicator (20min) prior to lipid extraction.Monophasic lipid extraction followed the method previously reported by Lydic et al. (Lydic et al., 2015) with modification as described below. 120 μl of MilliQwater, 420 μl of methanol with 0.01% (w/v) BHT, and 270 μl of chloroform were added to all samples. For every 10 μg protein present in the samples, 1 μl of a customised isotope labelled internal standard lipid mixture and 1 μl of a d5-TG Internal Standard Mixture I (Avanti Polar Lipids, Alabaster, AL, USA) were added. The customised isotope labelled internal standard mixture was comprised of 14 deuterated lipid standards (Avanti Polar Lipids, Alabaster, AL, USA): 15:0-18:1(d7) PC (250 μM), 15:0-18:1(d7) PE (240 μM), 15:0-18:1(d7) PS (250 μM), 15:0-18:1(d7) PG (20 μM), 15:0-18:1(d7) PI (220 μM), 15:0-18:1(d7) PA (180 μM), 18:1(d7) LPC (45 μM), 18:1(d7) LPE (10 μM), 18:1(d7) Chol Ester (10 μM), 18:1(d7) MG (10 μM), 15:0-18:1(d7) DG (17 μM), 18:1(d9) SM (80 μM), d18:1(d7)-15:0 Cer (40 μM) and Cholesterol(d7) (20 μM). The d5-TG Internal StandardMixture I contained 20:5-22:6-20:5 (d5) TG (4.03 μM), 14:0-16:1-14:0 (d5) TG(3.99 μM), 15:0-18:1-15:0 (d5) TG(3.97 μM), 16:0-18:0-16:0 (d5) TG(4.05 μM), 17:0-17:1-17:0 (d5) TG(4.14 μM), 19:0-12:0- 19:0 (d5) TG (4.01 μM), 20:0-20:1-20:0 (d5) TG (3.81 μM), 20:2-18:3-20:2 (d5) TG (3.96 μM), 20:4-18:2-20:4 (d5) TG (3.90 μM). The 15:0-18:1-15:0 (d5) TG was used for semi-quantification of endogenous TG lipids. Samples were vortexed thoroughly and incubated with 1,000 rpmshaking at room temperature for 30min, followed by centrifugation at 14,000 rpmat roomtemperature for 15 min. Supernatants containing lipids were transferred to new tubes. The remaining pellets were re-extracted with 100 μL of MilliQ water and 400 μl of chloroform:methanol (1:2, v:v) containing 0.01% (w/v) butylated hydroxytoluene (BHT) following incubation and centrifugation as described above. The supernatants from the repetitive extractions were collected and pooled, dried by evaporation under vacuum using a GeneVac miVac sample concentrator (SP Scientific, Warminster, PA, USA) and then reconstituted in isopropanol:methanol:chloroform (4:2:1, v:v:v, containing 0.01% BHT) at a final concentration of 4 μl lipid extract per μg protein. |
| Sample Derivatization: | Derivatization of aminophospholipids (i.e., PE and PS) and plasmalogen-containing lipids followed the method previously reported by Ryan and Reid (2016). Prior to derivatization, a 2.5 mM stock solution of triethyamine (TEA) in chloroform was freshly prepared by adding 3.4 μl TEA to 10 ml of chloroform. A 2.5 mM stock solution of S,S’- dimethylthiobutanoylhydroxysuccinimide ester iodide (13C1-DMBNHS) was freshly prepared by dissolving 4.87 mg 13C1- DMBNHS in 5 ml of dimethylformamide (DMF). A stock solution of 3.94 mM iodine was freshly prepared by dissolving 10 mg iodine in 10 ml chloroform. A stock solution of 90 mM ammonium bicarbonate was freshly prepared by dissolving 35.6 mg ammonium bicarbonate in 5 ml of HPLC methanol. A solution of 2:1 (v:v) chloroform:methanol containing 266 μM iodine and 2mMammoniumbicarbonatewas prepared by adding 160 μl of 3.94mMiodine in chloroformto 1.44ml chloroform, and 53.3 μl of 90mMammonium bicarbonate inmethanol to 746.7ml methanol, then combined and placed in an ice bath. Due to the limitations in sample amounts, no replicate derivatization reactions were performed. 4 μl of brain tissue or BDEV lipid extracts were aliquoted to individual wells of a Whatman Multi-Chem 96-well plate (Sigma Aldrich, St. Louis, MO, USA). The solvent was evaporated under vacuum with a GeneVac miVac sample concentrator. 40 μl of a solution of 39:1.1:1 (v:v:v) chloroform:2.5 mM TEA:2.5 mM 13C1-DMBNHS reagent was added to each dried lipid extract and the 96-well plate was sealed with Teflon Ultra Thin Sealing Tape. Samples were then incubated at room temperature with gentle shaking for 30 min. After incubation, the solvents were evaporated under vacuum with a GeneVac miVac sample concentrator and samples were chilled on ice for 10 min prior to addition of 40 μl of the 2:1 (v:v) chloroform:methanol containing 266 μMiodine and 2 mMammonium bicarbonate. Reactions were mixed by careful pipetting and the plate was sealed with aluminiumfoil and then placed on ice for 5min before solvents were completely removed by evaporation under vacuum with a GeneVac miVac sample concentrator. The dried lipid extracts were washed three times with 40 μl of 10 mM aqueous ammonium. Remaining traces of water were then removed by evaporation under vacuum with a GeneVac miVac sample concentrator. The derivatized brain tissue lipid extracts and BDEV lipid extracts were then resuspended in 50 μl and 25 μl of isopropanol:methanol:chloroform(4:2:1, v:v:v) containing 20mMammoniumformate respectively. The 96-well plate was then sealed with Teflon Ultra Thin Sealing Tape prior to mass spectrometry analysis. |
Combined analysis:
| Analysis ID | AN006187 | AN006188 | AN006189 |
|---|---|---|---|
| Analysis type | MS | MS | MS |
| Chromatography type | None (Direct infusion) | None (Direct infusion) | None (Direct infusion) |
| Chromatography system | none | none | none |
| Column | none | none | none |
| MS Type | ESI | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap | Orbitrap |
| MS instrument name | Thermo Orbitrap Fusion Lumos Tribrid | Thermo Orbitrap Fusion Lumos Tribrid | Thermo Orbitrap Fusion Lumos Tribrid |
| Ion Mode | POSITIVE | NEGATIVE | POSITIVE |
| Units | pmol/µg protein | pmol/µg protein | pmol/µg protein |
Chromatography:
| Chromatography ID: | CH004696 |
| Chromatography Summary: | Direct infusion lipidomic analyses were conducted. For underivatized samples, 4 μl of brain tissue or BDEV lipid extracts were aliquoted in triplicate to individual wells of a twin-tec 96-well plate (Eppendorf,Hamburg,Germany). The brain tissue lipid extracts andBDEVlipid extractswere dried and then resuspended in 50 μl (brain tissue) and 25 μl (BDEV) of isopropanol:methanol:chloroform(4:2:1, v:v:v) containing 20mMammonium formate respectively. The 96-well plate was then sealed with Teflon Ultra Thin Sealing Tape prior to mass spectrometry analysis. 10 μl of each underivatized or derivatized lipid sample was aspirated and introduced via nano-ESI to an Orbitrap Fusion Lumos mass spectrometer (ThermoFisher Scientific, San Jose, CA, USA) using anAdvion TriversaNanomate (Advion, Ithaca,NY, USA) operating with a spray voltage of 1.1 kV and a gas pressure of 0.3 psi in both positive and negative ionizationmodes. For MS analysis, the RF lens was set at 10%. Full scan mass spectra were acquired at a mass resolving power of 500,000 (at 200 m/z) across a m/z range of 350 – 1600 using quadrupole isolation, with an automatic gain control (AGC) target of 5e5. Themaximum injection time was set at 50 ms. Spectra were acquired and averaged for 3 min. Following initial ‘sum-composition’ lipid assignments by database analysis (see below), ‘targeted’ higher-energy collision induced dissociation (HCD-MS/MS) product ion spectra were acquired on selected precursor ions at amass resolving power of 120,000 and default activation times in positive ionizationmode using the underivatized lipid extracts to confirm the identities of lipid head groups, or in negative ionization mode using underivatized lipid extracts for fatty acid chain identification. HCD-MS/MS collision energies were individually optimized for each lipid class of interest using commercially available lipid standards whenever possible. |
| Instrument Name: | none |
| Column Name: | none |
| Column Temperature: | none |
| Flow Gradient: | none |
| Flow Rate: | none |
| Solvent A: | none |
| Solvent B: | none |
| Chromatography Type: | None (Direct infusion) |
MS:
| MS ID: | MS005891 |
| Analysis ID: | AN006187 |
| Instrument Name: | Thermo Orbitrap Fusion Lumos Tribrid |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Non derivatized lipids were measured in positive mode in triplicate. ‘Sum composition’ level lipid identifications were achieved using a developmental version of LipidSearch software 5.0α (Mitsui Knowledge Industry, Tokyo, Japan) by automated peak peaking and searching against a user-defined custom database of lipid species (including the deuterated internal standard lipid species and allowing for the mass shifts introduced by 13C1-DMBNHS and iodine/methanol derivatization). The parent tolerance was set at 3.0 ppm, a parent ion intensity threshold three times that of the experimentally observed instrument noise intensity, and a max isotope number of 1 (i.e., matching based on the monoisotopic ion and the M+1 isotope), a correlation threshold (%) of 0.3 and an isotope threshold (%) of 0.1. The lipid nomenclature used here follows that defined by the LIPID MAPS consortium (Fahy et al., 2005). Semi-quantification of the abundances of identified lipid species was performed using an in-house R script, by comparing the identified lipid ion peak areas to the peak areas of the internal standard for each lipid class or subclass, followed by normalization against the total protein amount in the samples. |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | Su_et_al_2021.pdf |
| MS ID: | MS005892 |
| Analysis ID: | AN006188 |
| Instrument Name: | Thermo Orbitrap Fusion Lumos Tribrid |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Non derivatized lipids were measured in negative mode in triplicate. ‘Sum composition’ level lipid identifications were achieved using a developmental version of LipidSearch software 5.0α (Mitsui Knowledge Industry, Tokyo, Japan) by automated peak peaking and searching against a user-defined custom database of lipid species (including the deuterated internal standard lipid species and allowing for the mass shifts introduced by 13C1-DMBNHS and iodine/methanol derivatization). The parent tolerance was set at 3.0 ppm, a parent ion intensity threshold three times that of the experimentally observed instrument noise intensity, and a max isotope number of 1 (i.e., matching based on the monoisotopic ion and the M+1 isotope), a correlation threshold (%) of 0.3 and an isotope threshold (%) of 0.1. The lipid nomenclature used here follows that defined by the LIPID MAPS consortium (Fahy et al., 2005). Semi-quantification of the abundances of identified lipid species was performed using an in-house R script, by comparing the identified lipid ion peak areas to the peak areas of the internal standard for each lipid class or subclass, followed by normalization against the total protein amount in the samples |
| Ion Mode: | NEGATIVE |
| Analysis Protocol File: | Su_et_al_2021.pdf |
| MS ID: | MS005893 |
| Analysis ID: | AN006189 |
| Instrument Name: | Thermo Orbitrap Fusion Lumos Tribrid |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Derivatized lipids were measured once in positive mode only. ‘Sum composition’ level lipid identifications were achieved using a developmental version of LipidSearch software 5.0α (Mitsui Knowledge Industry, Tokyo, Japan) by automated peak peaking and searching against a user-defined custom database of lipid species (including the deuterated internal standard lipid species and allowing for the mass shifts introduced by 13C1-DMBNHS and iodine/methanol derivatization). The parent tolerance was set at 3.0 ppm, a parent ion intensity threshold three times that of the experimentally observed instrument noise intensity, and a max isotope number of 1 (i.e., matching based on the monoisotopic ion and the M+1 isotope), a correlation threshold (%) of 0.3 and an isotope threshold (%) of 0.1. The lipid nomenclature used here follows that defined by the LIPID MAPS consortium (Fahy et al., 2005). Semi-quantification of the abundances of identified lipid species was performed using an in-house R script, by comparing the identified lipid ion peak areas to the peak areas of the internal standard for each lipid class or subclass, followed by normalization against the total protein amount in the samples |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | Su_et_al_2021.pdf |
