lipid analysis with differential mobility...
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2 © 2012 AB SCIEX
Lipids Fatty Acids Steroids
Lipid
Vitamins Terpenes
Eicosanoids
Glycerophospholipids
Triglycerides Waxes Sphingolipids
Ether Phospholipids
Platelet-
Activating
Factor
Plasmalogens Diacyl-Linked
Phospholipids
PC PE PS PI PG
Ceramides
Sphingomyelins
Cerebrosides
Gangliosides
Lipids, store of
metabolic energy,
with essential role
in human
physiology:
• Metabolic
homeostasis
• Cell signaling
• Cell and
organelle
structure
And disease:
• Inflammation
• Cancer
• Cardiovascular
disease
• Inflammatory
bowel diseases
• Neurological
diseases PA
The huge diversity of isomers and isobaric compounds confounds confident ID
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• There are as many as 180,000 different lipid molecular species that
are found in a narrow mass range of ~700 amu; most LC
applications require 2D separation or multiple injections
LipidView calculator exercise:
Select mass of 762.4 with a
tolerance of 0.1 amu
→ 25 Lipids identified
Considering the Q1 selection
window is ~1.2 Da, isobaric
overlap makes unambiguous
identification and quantification
nearly impossible
Challenges in Lipidomic Analysis
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Current Strategies in Lipidomics
Separation problem:
– LC chromatography
– Chiral chrom (Ecosaniods, up to 40 min)
– Solid Phase Extraction
– Fractionation
– Chemical deriviatization
Mass spectrometry approaches:
– Dedicated precursor ion and neutral loss scans (Qtrap® Systems)
– IDA-based methodologies (Triple TOF and Qtrap® Systems)
– MS/Msall (Triple TOF)
New approach: Differential Mobility Spectrometry
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How does DMS separate Ions?
Separation waveform (SV):
Alternating between low and high
fields radially displaces ions
towards one or the other
electrode, depending upon high
and low mobility characteristics
Compensation voltage (COV):
Small DC offset that 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
SV COV
To
MS Gas
flow
•Differential Mobility Spectrometry (DMS) is the term used for planar geometry
Dimentions:10x30x1mm
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Phospholipid (PL) Sub-Classes
Differential
charge site
topography
makes
phospholipids
ideal
candidates for
differential
separation by
SelexION DMS
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PC PI
PS PA
PE
PG
Isobaric Overlap of Phospholipids Experiment: EMS scan of Bovine Heart Extract (BHE)
• Lipidomic
spectra are
incredibly
complex
• MS/MS spectra
generated on
precursors in
zones of
isobaric overlap
will contain
product ions
form other
isobaric species
The limitations to lipidomic analysis are sample related (i.e., isobaric
overlap). SelexION is uniquely positioned to address this challenge because
it separates molecules PRIOR to MS analysis
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SelexIon Separates Phospholipid Standards
PC
PE
PA
PG
PS
PI • Using DMS alone, a
mixture of lipids can
be separated into it’s
individual
components
• Baseline separation
can be achieved,
completely
abrogating isobaric
interference
Experiment: MRM scan of 6 phospholipid standards with COV ramp
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: EMS Scan of Bovine Heart Extract; NO DMS
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PC
PC
PE
PE
PA
PA
PG
PG
PS
PS
PI
PI
Resolution of Phospholipids in a Biological Lipid Extract by DMS
Individual
phospholipid
classes can be
extracted from an
EMS scan based
on differential
COV
• Simplifies
Lipidview
searching
• Increases
confidence in
lipid
identification
Experiment: EMS scan of bovine heart extract with ramped COV
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DMS Resolves Phospholipids and Glycerolipids Experiment: EMS (POS) Scan of Bovine Heart Extract with COV ramp
Fast separation of neutral and polar lipids without chromatography
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DMS Resolves PC and Sphingolipids
Precursor Ions +184
Phospholipids Sphingomyelins
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MS/MS Spectrum with DMS OFF
18:0
18:2
Product ions of 786.5
Quantitation and Lipidomics • Accurate quantitation is problematic in top down lipidomics due to isobaric
interference: at a nominal mass of 786.5, there are 41 common lipid species
18:1
Experiment: EPI (-) scan of bovine heart extract (m/z 786.5); NO DMS
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DMS removes isobaric interference, enabling more
accurate quantitation and identification
PC 36:2 PS 36:3 *
* *
DMS and MS/MS Analysis Experiment: EPI scan of BHE (m/z 786.5) with COV ramp
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12(R)-HETE
5(R)-HETE
11(R)-HETE
12(S)-HETE
5(S)-HETE
11(S)-HETE
DMS Resolves Positional and R and S Isomers of
Hydroxy-eicosatetrenoate
Experiment: MRM scan of infused eicosanoid standard mix; ramped COV
• Oxidized
metabolites of
Archidonic acid
• No chiral
chromatography
for resolution
• Resolves a major
problem in
eicosanoid
analysis
ALL lipid classes we have evaluated are ‘DMS-active’
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Benefits of Using SelexION for Lipidomic Analysis
• Only SelexION 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
Effective lipid
resolution without
complicated
chromatographic
strategies
18 © 2012 AB SCIEX
Acknowledgements
• Paul RS Baker
• Larry Campbell
• Brigitte Simons
• Christie Hunter
Legal Acknowledgements
− 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.
− © 2013 AB SCIEX.
20 © 2012 AB SCIEX
Separations are Chemical in Nature
High field
Declustering
Mobility Increases
Low Field
Clustering-
Mobility Decreases
+
Krylov et al., Int. J. Mass Spectrom., 2009, 285, 149-156
High field
Declustering
Mobility Increases
Low Field
Clustering-
Mobility Decreases
Dynamic Cluster/Decluster Model