The Unique Nature of Lipids and How MS/MSALL Is
an Ideal Tool for Comprehensive Lipidomics
Jeff McDonald, Ph.D.
Department of Molecular Genetics
UT Southwestern Medical Center
Dallas, TX
June 18th, 2014
AB Sciex Breakfast Seminar
ASMS 2014
What is a LIPID?
1) “..hydrophobic or amphipathic small molecules that may originate
entirely or in part by carbanion-based condensations of thioesters
(fatty acids,polyketides, etc.) and/or by carbocation-based
condensations of isoprene units (prenols, sterols, etc.).”
2) “Compounds that partition to the organic layer of a two-phase BlighDyer extraction”
Bligh/Dyer Extraction
plasma
Aqueous (MeOH/H2O)
Organic (lipids) (DCM/MeOH)
A Brief Overview of Lipids
J Lipid Res, 51, 3299-3305 (2010)
Basic Structures of Glycerolipids
C18:0
SN1
SN3
C18:0
SN2
C18:0
• Triglyceride 18:0/18:0/18:0 (TAG)
– C54:0
• Fully saturated acyl chains
• Same acyl chain at SN 1, 2, and 3
position
Basic Structures of Glycerolipids
C18:0
C18:0
C18:0
SN1
SN1
SN3
SN3
SN2
C18:0
• 2,3 Diacylglycerol 18:0/18:0 (DAG)
SN2
• 1,3 Diacylglycerol 18:0/18:0 (DAG)
• 2 Fully saturated acyl chains
• Same acyl chain at SN 2 and 3 position
Basic Structures of Glycerolipids
C18:0
SN1
C18:0
SN2
SN3
C18:0
• Monoacylglycerol 18:0 (MAG)
• Fully saturated acyl chain at either SN1, 2, or 3
The Complexity of Glycerolipids
C16:0
C18:0
C20:0
SN1
SN3
SN2
• 16:0/18:0/20:0 C54:0 TAG
• Acyl chains can vary from ~10-24 carbons
– Almost entirely even number species
• 16:0 SN1, 18:0 SN3, 20:0 SN2
• Same for DAGs and MAGs
The Complexity of Glycerolipids
C16:1
SN1
SN3
C18:1
SN2
C20:1
• 16:1/18:1/20:1 C54:3 TAG
• Omega 3 (SN1), 6 (SN3), 9 (SN2)
– Double bonds can be anywhere in the acyl
chain
– Can have multiple double bonds in acyl chain
• Same for DAGs and MAGs
The Complexity of Glycerolipids
C16:1
SN1
SN3
C18:1
SN2
C20:1
• Basic characteristics of neutral glycerolipids
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–
–
–
Acyl chain length
Number and position of double bonds
SN positions
TAG, DAG, MAG
• Advanced characteristics of neutral glycerolipids
– Ether linked fatty acyl groups
– Oxidized fatty acids
– Cis/trans double bond configurations
Add a Unique Head Group to Make Phospholipids
SN3
C18:0
C20:4
SN1
SN2
• 18:0-20:4 Phosphotidylcholine
– Possible head groups
•
•
•
•
•
•
Choline
Ethanolamine
Phosphatidic acid
Glycerol
Serine
Inositol
– Other variables
•
•
•
•
Ether linked fatty acids
Lyso confrigurations
Plasmalogens
Oxidized fatty acids
The Complexity of Acyl Lipids
Solubility/Polarity between and within lipid classes
require unique analytical approaches
H2O
Acyl Coenzyme A
MeOH
Phospholipids
DCM
Triglycerides
Bad News:
• Unique extractions
• Unique chromatography
• Very few defined standards available
Good News:
• Common mass spectrometry
Cholesteryl esters
Mass Spectrometry of Glycerolipids
C18:1
C18:1
C18:1
•
•
•
•
m/z 902 (M+NH4) positive mode
Acyl chain configuration = C54:3
18:1/18:1/18:1 is one possibility
Numerous other isomers possible
–
–
–
•
•
16:1/18:1/20:1
16:0/18:0/20:3
16:0/16:0/22:3
CID results in neutral loss of one fatty acid (with
ammonium adduct)
One “molecular ion” may show many fatty acid losses
One m/z (TAG) Shows Diverse Fatty Acid Profile
M-18:1
Mouse liver extract
Product ion m/z 902 Da
NL 299
18:1
M-18:2
M-18:0
M+NH4
M-20:1
M-16:0
The Mass Spectrometry of Phosphotidylcholine
•
•
•
•
•
•
m/z 810 (M+H) in positive mode
Acyl chain configuration = C38:4
18:0/20:4 is one possibility
Numerous other isomers possible
Collision induced dissociation results in loss of choline
head group m/z 184
No specific fatty acid information in positive mode
Phosphotidylcholines Show Common Fragment Ion
Choline head group
m/z 184
Product ion m/z 810
Mouse liver extract
M+H
The Workhorses of Lipid Analysis
AB Sciex Triple Quadrupoles
Targeted Quantitative Analysis: A Narrow
View of the World
Infusion-Based MS: The Whole World From
Any Angle
Lipidome of Human Plasma
NIST SRM
1950
588
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
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
Ursus Analyticus
Infusion-Based MS Platform for Lipidomic Analysis
1) Sample Extraction
(CH2Cl2/MeOH)
20uL human plasma
Bulk lipid extract
(DCM:MeOH:IPA)
Insoluble material/
proteins
LEAP Robotic Infusion System
AB SCIEX TripleTOF 5600+
Workflow for Infusion-Based Global Lipidomics
1
2
B/D Extraction
of plasma
Infuse into
MS
m/z 369
Plasma+Stds
Acquire TOF
Spectrum
Product-ion
Spectrum
CE
m/z 666
30 minutes
Aqueous
4
3
Organic (lipids)
Scan 200-1200 Da
30-40K resolution
< 2ppm mass accuracy
Hour Time Scale
PC
m/z 666
Cholesterol
PE
DG
SM
Acquire MS/MSALL Data
Analysis
Spectrum
m/z 666
TOF 8Spectrum
CE 18:2
minutes
Neutral Loss 279 = FA 18:2
TG
5
Mining Mouse Liver Dataset Post-Acquisition
MS/MSALL identifies 1000’s of MS features
Lipids that contain 18:2 fatty acid
Neutral-loss 297 Da
Triacylglycerol
Cholesteryl Ester
Lipids that contain choline head group
Precursor 184 Da
Lyso phosphatidylcholines
Phosphatidylcholines
Mining Mouse Liver Dataset Post-Acquisition
Triacylglycerol
Neutral Loss 297 (FA 18:2)
Cholesteryl Ester
Triacylglycerol
Neutral Loss 299 (FA 18:1)
Cholesteryl Ester
Triacylglycerol
Neutral Loss 301 (FA 18:0)
Cholesteryl Ester
PCA Analysis – MS/MSALL Data
Liver
Plasma
Blank
MarkerView (AB Sciex)
Case-Study on Fatty Liver Disease
Hepatic Steatosis: accumulation of triglycerides in
hepatocyte lipid droplets
J C Cohen et al. Science 2011;332:1519-1523
Experiment Design
• 4 groups of mice:
– LXR knockout (high fat diet, high fat diet + ezetamibe)
– Wild-type (high fat diet, high fat diet + ezetamibe)
• Collect livers
• Measure total triglycerides and cholesterol
– Done by “TG Kit” and by GC-FID
• GC-FID method dates back to 60’s
Figure courtesy of Joyce Repa
Liver Triglyceride Concentrations*
* Measured by kit-based assay
Figure courtesy of Joyce Repa
Liver Lipids analysis with MS/MSALL
WT HFD EZ
WT HF
KO HFD
KO HFD EZ
Liver Lipid analysis with MS/MSALL
TAG C50
18:2
WT HFD
KO HFD
WT HFD EZ
KO HFD EZ
WT HFD EZ
KO HFD EZ
TAG C54
18:2
WT HFD
KO HFD
The Future of MS/MSALL Lipidomics
• Evaluate sample fractionation to improve “coverage” of lipidome
– Liquid-liquid, SLE or SPE, off-line HPLC – fraction collection
• Implement SelexION Ion Mobility
– Resolve isobaric species
• OZid
– Determine double bond positions
– Blanksby, University of Wallongong
• Continue working with Sciex on software and data analysis tools
– Build set-lists into MarkerView
– Link Markerview/MultiQuant to LIPIDVIEW
• Currently developing custom software programs for data
reduction and interpretation
Acknowledgements
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•
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LIPID MAPS community
David Russell – UTSW
Phil Sanders – Eli Lilly
AB Sciex
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Brigitte Simons
Jennifer Krone
Mark Cafazzo
Lyle Burton
• AB-Sciex Academic Partnership Program
• Joyce Repa
• NIH, Clayton Foundation for Research