The LipidyzerA New Platform for Quantitative Lipidomics
Cyrus Papan
Sr. Support Specialist, SCIEX
Munich, 11. May 2016
The Challenge in Lipidomics Research
‒Lipids are polymeric structures
and their individual elements
have their own pathways
‒Breakdown into intermediary
metabolites (FAs)
‒Mapping lipids requires mapping
to the right level
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Intermediary
Complex
Aggregates
FAs
TAGs
VLDL
Fatty acid
metabolism
Acyltransferase
metabolism
Lipid class
metabolism
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What is needed from a Lipid Platform
1) Specificity
1
‒ A non-specific method (e.g. PC 36:2)
does not allow mapping to the
elements of the matrix
2) Quantitation
2
‒ A non-quantitative approach does
not allow accurate summing of the
rows and columns
3) Comprehensive Coverage
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‒ A partially complete matrix is difficult
to interpret
Metabolon®
Global Leader in Metabolomics Applications
• Over 10 years of continuous leadership in
metabolomics technology development
• Core business is metabolomics services
and diagnostic development
• Over 4000 projects conducted with
hundreds of clients
• 500 publications, many in top tier journals
(Nature, Science, Cell)
• Acquired Lipomics in 2012, a lipidomics
company founded in 2000
Partnered with SCIEX to build Next-Gen Lipidomic capabilities
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Simplifying the Complexity
Powered by METABOLON®
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Why the Lipidyzer™ Platform?
Standardization of Sample Preparation
• Novel internal standard kits and methods
designed exclusively for the Lipidizer™.
• Built on Metabolon’s “know-how” of
commercial lipid analysis platforms and
standard procedures
• Provides user with confident, reproducible
quantitation of plasma and serum samples
• Over 50 internal standards across ten
lipid classes – a complete unique
strategy!
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How the LipidyzerTM Platform Works
LipidyzerTM Enables Ease of Use, Specificity and Quantitative Rigor
Three Key Elements of the Platform:
1. Software: Ease of Use
•
•
Log samples, create batches
Automated calculation of chemistries, tuning and system tests
2. SelexIONTM: Specificity
• Resolve isobaric interference between different lipid classes
• Determine lipid class and molecular species composition in a
single run
3. Internal Standards: Quantitation
• Ensure spray stability
• Minimize carryover
• Neutralize quantitative bias
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LipidyzerTM Enables Ease of Use, Specificity and Quantitative Rigor
Three Key Elements of the Platform:
1. Software: Ease of Use
•
•
Log samples, create batches
Automated calculation of chemistries, tuning and system tests
2. SelexIONTM: Specificity
• Resolve isobaric interference between different lipid classes
• Determine lipid class and molecular species composition in a
single run
3. Internal Standards: Quantitation
• Ensure spray stability
• Minimize carryover
• Neutralize quantitative bias
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Lipidomics Workflow Manager: Dashboard
Simple and Guided Workflow
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Lipidomics Workflow Manager: Dashboard
System Suitability Tests and Automated Tuning for SelexION
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Lipidomics Workflow Manager: Sample Login and Metadata Entry
Flexible Import
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Lipidomics Workflow Manager: Internal Standard Assembler
Information from Registered Kits
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Lipidomics Workflow Manager: Data Visualization
Raw data
Heat Maps and Statistics
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LipidyzerTM Enables Ease of Use, Specificity and Quantitative Rigor
Three Key Elements of the Platform:
1. Software: Ease of Use
•
•
Log samples, create batches
Automated calculation of chemistries, tuning and system tests
2. SelexIONTM: Specificity
• Resolve isobaric interference between different lipid classes
• Determine lipid class and molecular species composition in a
single run
3. Internal Standards: Quantitation
• Ensure spray stability
• Minimize carryover
• Neutralize quantitative bias
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Lipidyzer™ Hardware Configuration
QTRAP® 5500 System with SelexION™ Technology
• Differential Mobility Spectrometry (DMS)
• Installation / removal of DMS in < 2mins – no
tools required
Source
Extension
Ring
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Curtain
Plate
DMS
cell
Differential Mobility Spectrometry (DMS)
SelexION™ Technology
• Planar geometry
• Gas flow towards MS draws ions
Gas
flow
To
MS
SV
COV
Waveform
DMS Dimensions
1x10x30 mm
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(transport gas)
• Asymmetric waveform applied
which alternates between high
field, K(E) and low field, K(0) –
separation voltage (SV)
‒ Moves charged ion back and forth
between plates
‒ Ion will have net drift base on its high
and low field mobility
• Compensation voltage (COV) is
small DC offset between the
plates – filtering voltage
Separation of Lipid Classes Using SelexIONTM
Negative Ion Mode
LPC
LPE
PC
PE
SM
100%
80%
60%
40%
20%
0%
-18
-16
-14
-12
-10
-8
-6
-4
COV (V)
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-2
0
2
4
6
8
PC/PE Standard Mixture Resolution
SelexIONTM Technology effectively resolves different lipid classes
DR=7
DR=15
DR=30
PC+PE
PC
PE
36
Resolution gas (DR) slows down ions in the DMS cell to enhance resolution
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Sample Carry Over Between Injections
Use of PEEKSIL tubing
1st Injection
• The use of PEEKSIL
5e7
Spectrum during elution
1.2e5
Spectrum during washing
tubing dramatically
reduces carryover
compared to regular
PEEK tubing
• A wash step at the end of
acquisition is enabled
into the method by
ramping the flow rate up
to 30µl/min
• Background level
3rd Injection
reduced to very low level
in the same run
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1.3e4
Spectrum of the 2nd
consecutive blank injection
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LipidyzerTM Enables Ease of Use, Specificity and Quantitative Rigor
Three Key Elements of the Platform:
1. Software: Ease of Use
•
•
Log samples, create batches
Automated calculation of chemistries, tuning and system tests
2. SelexIONTM: Specificity
• Resolve isobaric interference between different lipid classes
• Determine lipid class and molecular species composition in a
single run
3. Internal Standards: Quantitation
• Ensure spray stability
• Minimize carryover
• Neutralize quantitative bias
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Unequal Fragmentation Efficiency of Lipids
Diversity of Fatty Acid Chain Lengths and Degrees of Unsaturation Result in
Differential Fragmentation Efficiency which Impacts Quantitation
Murphy et al. Chem Rev 2001, 101, 479-526
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A Broad Range of Internal Standards to Normalize Quantitative Data
Multiple Internal Standards that Reflect the Diversity of Lipid Molecular Species
Each lipid class has multiple internal standards at concentrations that reflect
those found in biology
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The LipidyzerTM Eliminates Quantitative Bias
Multiple internal standards per class provide accurate quantitation
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The LipidyzerTM Generates Accurate Lipid Class Quantitation
Quantitative data with < 10% bias and ~ 5% RSD for lipid classes
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Conclusions
Three Key Elements of the Platform:
1. Software: Ease of Use
•
•
Log samples, create batches
Automated calculation of chemistries, tuning and system tests
2. SelexION: Specificity
• Resolve isobaric interference between different lipid classes
• Determine lipid class and molecular species composition in a
single run
3. Internal Standards: Quantitation
• Ensure spray stability
• Minimize carryover
• Neutralize quantitative bias
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Acknowledgements
Sciex
METABOLON
Avanti Polar Lipids
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Baljit Ubhi
Paul Baker
Eva Duchoslav
Larry Campbell
Leo Wang
Pauline Vollmerhaus
Aaron Hudson
Mark Cafazzo
Marjorie Minkoff
Alex Conner
Steve Watkins
Annie Evans
Richard Robinson
Joseph Yu
Luke Miller
Sarada Tanikella
Peter Zhu
Corey DeHaven
Walt Shaw
Lisa Connelly
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.
© 2015 AB SCIEX.
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