ANSWERS FOR SCIENCE. KNOWLEDGE FOR LIFE.
SelexION™ Technology for Lipid Analysis:
Pushing the Boundaries of Lipidomics
Baljit Ubhi, Ph.D
ASMS Baltimore, June 2014
Lipidomics Profiling Needs and Deliverables
Liver metabolism
Ståhlman, M. et al J Chromatogr B Analyt Technol Biomed Life Sci. 2009
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© 2014 AB SCIEX
The Lipidome
Comprised of Multiple, Distinct Structural Lipid Classes
Fatty Acids Steroids
Lipid
Vitamins
Terpenes
Lipids play an
essential role in
human physiology:
•
Eicosanoids/
Oxidized FA
Mono-, Di- and Waxes
Triglycerides
Glycerophospholipids
Ether Phospholipids
Sphingolipids
Sphingomyelins
Ceramides
Cerebrosides
Diacyl-Linked
Phospholipids
Gangliosides
PlateletActivating Plasmalogens
Factor
Oxidized Phospholipids
Halogenated Lipids
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•
•
Metabolic
homeostasis
Cell signaling
Cell and organelle
structure
And disease:
•
•
•
•
•
•
Inflammation
Cancer
Cardiovascular
disease
Diabetes
Inflammatory
bowel disease
Neurological
diseases
Isobaric Overlap of Phospholipids
Experiment: EMS scan of Bovine Heart Extract (BHE)
• Lipidomic
spectra are
incredibly
complex
PC
PE
PA
PG
PS
PI
550
600
650
700
750
800
Mass/Charge (Da)
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850
900
950
• MS/MS spectra
generated on
precursors in
zones of
isobaric
overlap will
contain
product ions
from other
isobaric
species
SelexION™ Technology Ion Mobility Device Components
1. Orifice Plate
2. Ion Mobility Cell
• Robust, easy-to-install, hardware components:
‒
‒
‒
‒
5
No tools required
No cables
No need to break vacuum
Installation in about 2 minutes
© 2014 AB SCIEX
3. Curtain Plate
How Does DMS Separate Ions?
Differential Mobility Spectrometry (DMS) is the term used for planar geometry
To
MS
Gas/Modifier
flow
SV
Separation waveform (SV):
radially displaces ions towards
one or the other electrode,
depending upon high and
low mobility characteristics
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COV
Compensation voltage (COV):
restores the trajectory for a given
ion or range of ions to allow them
to transmit through the DMS device
and enter the mass spectrometer
SelexIONTM Technology Separates Phospholipid Classes
Experiment: MRM Scan of 6 Phospholipid Standards with COV Ramp
PI
PG
PS
PE
PC
PA
• Using DMS alone, a
mixture of lipids
can be separated
into it’s individual
components
• Baseline separation
can be achieved,
completely
abrogating isobaric
interference
Proof of concept: DMS separates simple lipid mixtures
Implications: “Cleaner” quantitative data using mrm scan modes (esp., MRM
HR); Improved qualitative analysis
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Effects of DMS on Complex Lipid Analysis
Can DMS help resolve lipids from a complex, biological lipid mixture?
Experiment 1: EMS Scan of Bovine Heart Extract; No DMS
723.70
7e6
6e6
5e6
529.48
4e6
3e6
747.41
766.42
861.36 885.33
2e6
698.46
1e6
480.32
450
500
786.39
794.38
592.54
550
600
650
700
750
800
850
Mass/Charge (Da)
Experiment 2: EMS Scan of Bovine Heart Extract; DMS with COV Ramp
8.3
-1.5
2.7
0.7
5.8
-5.2
-18 -16 -14 -12 -10 -8
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© 2014 AB SCIEX
-6
-4
-2
0
2
4
COV (V)
6
8
10 12 14 16 18
900
950
Resolution of Phospholipid Sub-Classes in a Biological
Lipid Extract by DMS
Experiment: EMS scan of bovine heart extract with ramped COV
EMS TIC
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An individual
phospholipid subclass can be
extracted from an
EMS scan based
on its differential
COV
•
Simplifies
LipidView™
Software
searching
•
Increases
confidence in
lipid
identification
Relationship Between Dipole Moment and COV
8
PI 16:0_16:0 [-H-]
6
4
PS 16:0_18:1 [-H-]
CV (V)
2
PG 16:0_18:1 [-H-]
0
-2
PE 16:0_18:1 [-H-]
-4
PA 16:0_18:1 [-H-]
-6
PC 16:0_18:1 [-H +Cl-]
-8
3
3.5
4
4.5
5
Model headgroup dipole moment (D)
Isopropanol as a chemical modifier in DMS
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© 2014 AB SCIEX
5.5
6
Lipid Analysis by AB SCIEX TripleTOF® 5600+ System
• Mass Range
‒ Q1 selection – 5 - 1250 m/z
‒ TOF MS – 5 – 40 000 m/z
• Speed
‒ 10 ms minimum accumulation times
‒ Up to 100 MS/MS scans per second in IDA
• Resolution
‒ High resolution mode ~30,000
‒ High sensitivity mode ~15,000
• Mass Accuracy
‒ 1-2 ppm RMS (external calibration)
• Dynamic Range
‒ ~ 3-4 orders
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TOF MS Analysis of Lung Lipid Extract Using DMS
Phospholipids; Cardiolipin
Fatty Acids
Experiment: TOF MS with COV ramp in negative ion mode;
Infusion of 100 μg/mL lung lipid extract
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Fatty Acid Resolution by DMS
1
Fatty Acids
1
2
3
3
2
Experiment: TOF MS with COV ramp in negative ion mode;
Infusion of 100 μg/mL lung lipid extract
Extracted Ion Chromatogram
16:1
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18:2
16:0
18:1
18:0
DMS Resolves Fatty Acid Cis/Trans Isomers
Experiment: TOF MS with COV ramp in negative ion mode;
Infusion of 100 μg/mL lung lipid extract
18:1 (cis)
16:1 (cis)
18:1 (trans)
16:1 (trans)
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Resolution of
cis/trans isomers
usually requires
chromatographic
separation prior to
analysis; DMS can
remove this need,
increasing the speed
of FA analysis
Resolution of Phosphatidylcholine (PC)
770.5307
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© 2014 AB SCIEX
792.5758
830.5917
854.5901
Resolution of Phosphatidylethanolamine (PE)
XIC 750.5446
PE 38.5
0.4 ppm for both
molecules
PC O-30:0 +AcO
PC O-30:0 + AcO is iso-elemental with PE 38:5
C43H77O7NP
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Resolution of Cardiolipin (ES(-))
Cardiolipin 74:5
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Resolution of Triglycerides from Complex Lipid Extracts
TAG 54:4+NH4
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Resolution of Triglycerides from Complex Mixtures of
Lipids
COV for TAG
COV Range 0.1 V
COV Range 1V
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SLICE: Structural Lipidomics Investigations using
Chemical Effects
• Molecules are
separated in CV
space prior to
entering the MS
• Chemical modifiers
enhance compound
resolution
DMS Separation
In CV Space
DMS-Separated Precursors
• Minimizes isobaric
overlap
MS/MS of
Selected Precursors
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Confident Identification of Isobaric Compounds
0.2u
Experiment: DMS MS/MSALL (NEG) of bovine heart extract
PE p16:0_22:5
PC p16:0_18:2
PE p16:1_22:4
CL 80:11
(20:4, 20:5, 18:1)
PG 18:1_18:0*
* 1st isotope
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© 2014 AB SCIEX Duchoslav E., Campbell, J. L., Baker, P.R.S., Patterson B.T., ASMS 2013, ThP 28-586
DMS Separation Improves Coverage and Confidence in
Lipid Species Annotation
Sample type
Acquisition strategy
TOF MS
Bovine Heart
DMS
TOF MS
E. Coli
DMS
Polarity
+
combined
+
combined
+
both
+
combined
# lipids*
135
225
266
248
340
466
164
138
174
81
167
201
Comparison of lipid profiles obtained with and without DMS separation
* Species with 3 or more chains (TAG, CL) are represented as sum of all
permutations
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SelexION™ Technology for Lipidomic Analysis
Effective lipid
resolution without
a need for
complicated
chromatographic
strategies
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© 2014 AB SCIEX
• SelexION™ Technology
isolates lipid classes prior to
MS/MS analysis, which
reduces isobaric and isotope
overlap and enables
accurate identification and
quantitation
• Lower LOQ/LOD for targeted
lipidomics; reduction in
matrix interference
Acknowledgements
Zora Biosciences
• Kim Ekroos
• Tuulia Sylvanne
UCSD
• Ed Dennis
• Oswald Quehenberger
• Paul Norris
• Aaron Armando
Wollongong University
Eli Lilly
• Todd Mitchell
• Steven Blanksby
• Simon Brown
• Al Maccarone
• Phil Sanders
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© 2014 AB SCIEX
AB SCIEX
• Paul Baker
• Larry Campbell
• Michael Jarvis
• Eva Duchoslav
• Brigitte Simons
• Christie Hunter
• Baljit Ubhi
Avanti Polar Lipids
LipidMAPS.org
Trademarks/Licensing
For Research Use Only. Not for use in diagnostic procedures.
The trademarks mentioned herein are the property of AB Sciex Pte. Ltd.
or their respective owners. AB SCIEX™ is being used under license. All
rights reserved. Information subject to change without notice.
© 2014 AB SCIEX.
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