Lipid analysis by multiple "omic" mass
spectrometry platforms
Where we’ve been, where we are, and where we’re going
Jeff McDonald
Department of Molecular Genetics
UT Southwestern Medical Center
Dallas, TX
LIPID MAPS “Glue Grant” NIH U54 GM 69338
Lipidome of Human Plasma
588
Lipids in Human Serum
Wikipedia : Reference ranges for blood tests - by mass.svg
pg/mL
mg/mL
Total cholesterol ~ 2 mg/mL
> 6 orders of
magnitude
Trace oxysterols < 2 ng/mL
Triglycerides ~ 1 mg/mL
Phospholipids ~ 2 mg/mL
Sphingolipids ~ 0.5 mg/mL
mg/mL
Cholesterol
Cholesteryl ester
General Steps of Lipid Extraction and Purification
1) Solvent extraction (DCM/MeOH)
2) Base hydrolysis
200μL serum + standards
KOH, 60°C
Organic-soluble
(lipids)
Insoluble material/
proteins
4) Solid phase extraction
Condition With Load sample Hexane
in hexane
rinse
hexane
3) Liquid-Liquid Extraction
23:1
CHCl3:MeOH
5) LC-MS
Aqueous (MeOH/H2O)
Organic (lipids) (DCM/MeOH)
Non polar
lipids
Sterols +
oxysterols
Analytical Separation and Detection of Lipids
HPLC
• Normal Phase
• Reverse Phase
• HELIC
GC
• DB5-MS 40m 0.18mm i.d.
o H2 carrier gas
• DB5-MS 30m 0.25
o He Carrier gas
Mass Spectrometry
• Triple Quadrupole (MRM)
• Neutral Loss Scan
• Precursor Ion Scan
Mass Spectrometry
• Electron Ionization
• Electron-Capture Negative
Ionization
o Methane
Measurement of the Plasma Lipidome
Sterols/Oxysterol
Neutral Lipids
3 Samples
2 Extractions
1 LC, 3 MS Analysis
Phospholipids
2 Samples
2 Extractions
2 LC-MS Analysis
Sphingolipids
1 Sample
3 Extractions
5 LC-MS Analysis
4 Samples
4 Extractions
2 LC-MS Analysis
2 GC-MS Analysis
Prenols/Cardiolipins
3 Samples
1 Extraction
3 LC-MS Analysis
Fatty Acids/
Eicosanoids
Human
Plasma
Lipidome
2 Samples
2 Extractions
1 LC-MS Analysis
1 GC-MS Analysis
Summary
15 Samples
14 Extractions
14 LC-MS methods
3 GC-MS methods
100’s standards
7 Expert Centers
$ Millions/many hours
The Workhorses of Lipid Analysis
AB Sciex Triple Quadrupole
Mass Spectrometers
Agilent GC-MS
Analytical Standards Generated by Avanti Polar Lipids
• >650 analytical lipid standards
–300 new lipid standards
–Primary, deuterated, odd-chain
• Purity and stability
–Packaging, storage, shelf-life
–In-house analytical expertise
Eicosanoids and Fatty Acids from Cayman Chemical
100+ eicosanoid
MS-Based Methods Developed by LM Cores
lipidmaps.org
A Ten-Year Timeline of Lipidomics
2013
2003
LIPID MAPS pioneers and advances speciated, quantitative LC/GC-MS methods for lipids
“Shotgun” Lipidomics introduced and implemented by Hann and Gross
Broad-scale, targeted and untargeted LC-MS “Omics” approaches flourish
Suizdak, Fiehn, Sumner, Newgard, Roddy, etc
Advanced, infusion-based
lipidomic methods introduced
One Sample, Many Choices
Sterols
Sphingolipids
Phospholipids
Neutral Lipids
Prenols
Eicosanoids/Fatty Acids
One Sample, Many Choices
“Ursus Analyticus” – Thanks Avanti!
Infusion-Based MS Platform for Lipidomic Analysis
1) Sample Extraction
(CH2Cl2/MeOH)
20uL human plasma
Small molecule
metabolome
Insoluble material/
proteins
LEAP Robotic Infusion System
AB SCIEX TripleTOF 5600+
MS with SelexION
Workflow for Infusion-Based Global Lipidomics
1
2
B/D Extraction
of plasma
Infuse into
MS
3
Acquire TOF
Spectrum
4
5
Acquire MS/MSALL Data
Analysis
Spectrum
Workflow for Infusion-Based Global Lipidomics
1
2
B/D Extraction
of plasma
Infuse into
MS
Plasma+Stds
Aqueous
Organic (lipids)
3
Acquire TOF
Spectrum
4
5
Acquire MS/MSALL Data
Analysis
Spectrum
Workflow for Infusion-Based Global Lipidomics
1
2
B/D Extraction
of plasma
Infuse into
MS
3
Acquire TOF
Spectrum
4
5
Acquire MS/MSALL Data
Analysis
Spectrum
Workflow for Infusion-Based Global Lipidomics
1
2
B/D Extraction
of plasma
Infuse into
MS
4
3
Acquire TOF
Spectrum
Acquire MS/MSALL Data
Analysis
Spectrum
TOF Spectrum
CE
Scan 200-1200 Da
30-40K resolution
< 2ppm mass accuracy
PC
Cholesterol
PE
DG
SM
TG
5
Workflow for Infusion-Based Global Lipidomics
1
2
B/D Extraction
of plasma
Infuse into
MS
4
3
Acquire TOF
Spectrum
5
Acquire MS/MSALL Data
Analysis
Spectrum
m/z 666
CE 18:2
Workflow for Infusion-Based Global Lipidomics
1
2
B/D Extraction
of plasma
Infuse into
MS
m/z 369
Acquire TOF
Spectrum
Product-ion Spectrum
m/z 666
Neutral Loss 379 = FA 18:2
m/z 666
4
3
5
Acquire MS/MSALL Data
Analysis
Spectrum
m/z 666
CE 18:2
Workflow for Infusion-Based Global Lipidomics
1
2
B/D Extraction
of plasma
Infuse into
MS
4
3
Acquire TOF
Spectrum
30 minutes
Acquire MS/MSALL Data
Analysis
Spectrum
5 minutes
Hour Time Scale
5
Mining the Dataset Post-Acquisition
MS/MSALL identifies 800+ MS features
Triacylglycerol with FA 18:1
Lipids that contain a 18:1 fatty acid
Cholesteryl ester C18:1
(NL 281Da)
Phosphatidylcholine
Lipids that contain a PC head group
(Prec 184 Da)
Lyso Phosphatidylcholines
Phosphatidylethanolamine
Lipids that contain a PE head group
(NL 141 Da)
Lyso Phosphatidylethanolamine
PCA Analysis – MS/MSALL Data
Liver
Plasma
Blank
MarkerView (AB Sciex)
Can Infusion-Based Metabolomics be Quantitative?
TAG 54:3
LPC 18:0
C 36:2 DAG
The Future of Lipidomics
• Chromatograhic-based quantitative analysis
–
–
–
–
HPLC resolving power and efficiency
MS sensitivity
MS acquisition speed
Flexible, parallel acquisition
• Infusion-based analysis
– Resolution, mass accuracy, linearity
– Data analysis software
– Off-line HPLC followed by infusion-based MS
• Ion-mobility
• Accessibility to standards
– Class-specific mixtures (primary and internal)
Acknolwedgements
• LIPID MAPS community
• Phil Sanders – Eli Lilly
• Brigitte Simons – AB Sciex
– Jennifer Krone
– Mark Cafazzo
– Lyle Burton
• AB-Sciex Academic Partnership Program