the lipidyzer- a new platform for quantitative … new platform for quantitative lipidomics cyrus...
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The Lipidyzer- A New Platform for Quantitative Lipidomics
Cyrus Papan
Sr. Support Specialist, SCIEX
Munich, 11. May 2016
3 © 2015 AB Sciex
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
Intermediary Complex Aggregates
FAs TAGs VLDL
Fatty acid
metabolism
Acyltransferase
metabolism
Lipid class
metabolism
7 © 2015 AB Sciex
What is needed from a Lipid Platform
1) Specificity ‒ A non-specific method (e.g. PC 36:2)
does not allow mapping to the
elements of the matrix
2) Quantitation ‒ A non-quantitative approach does
not allow accurate summing of the
rows and columns
3) Comprehensive Coverage ‒ A partially complete matrix is difficult
to interpret
1
2
3
8 © 2015 AB Sciex
Metabolon®
• 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
Global Leader in Metabolomics Applications
Partnered with SCIEX to build Next-Gen Lipidomic capabilities
10 © 2015 AB Sciex
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!
21 © 2015 AB Sciex
LipidyzerTM Enables Ease of Use, Specificity and Quantitative Rigor
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
Three Key Elements of the Platform:
22 © 2015 AB Sciex
LipidyzerTM Enables Ease of Use, Specificity and Quantitative Rigor
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
Three Key Elements of the Platform:
24 © 2015 AB Sciex
Lipidomics Workflow Manager: Dashboard
System Suitability Tests and Automated Tuning for SelexION
26 © 2015 AB Sciex
Lipidomics Workflow Manager: Internal Standard Assembler
Information from Registered Kits
27 © 2015 AB Sciex
Lipidomics Workflow Manager: Data Visualization
Raw data
Heat Maps and Statistics
28 © 2015 AB Sciex
LipidyzerTM Enables Ease of Use, Specificity and Quantitative Rigor
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
Three Key Elements of the Platform:
30 © 2015 AB Sciex
Lipidyzer™ Hardware Configuration
• Differential Mobility Spectrometry (DMS)
• Installation / removal of DMS in < 2mins – no
tools required
QTRAP® 5500 System with SelexION™ Technology
DMS
cell Curtain
Plate Source
Extension
Ring
31 © 2015 AB Sciex
SV COV
Gas
flow To
MS
Differential Mobility Spectrometry (DMS)
• Planar geometry
• Gas flow towards MS draws ions
(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
SelexION™ Technology
Waveform
DMS Dimensions
1x10x30 mm
33 © 2015 AB Sciex
Separation of Lipid Classes Using SelexIONTM
Negative Ion Mode
-18 -16 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8
COV (V)
0%
20%
40%
60%
80%
100%
LPC LPE PE PC SM
36 © 2015 AB Sciex
SelexIONTM Technology effectively resolves different lipid classes
DR=7 DR=15 DR=30
PC+PE
PC
PE
Resolution gas (DR) slows down ions in the DMS cell to enhance resolution
PC/PE Standard Mixture Resolution
39 © 2015 AB Sciex
Sample Carry Over Between Injections
Use of PEEKSIL tubing
Spectrum during elution
Spectrum during washing
Spectrum of the 2nd
consecutive blank injection
5e7
1.2e5
1.3e4
1st I
nje
ctio
n
3rd
Inje
ction
• The use of PEEKSIL
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
reduced to very low level
in the same run
40 © 2015 AB Sciex
LipidyzerTM Enables Ease of Use, Specificity and Quantitative Rigor
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
Three Key Elements of the Platform:
41 © 2015 AB Sciex
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
42 © 2015 AB Sciex
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
43 © 2015 AB Sciex
The LipidyzerTM Eliminates Quantitative Bias
Multiple internal standards per class provide accurate quantitation
44 © 2015 AB Sciex
The LipidyzerTM Generates Accurate Lipid Class Quantitation
Quantitative data with < 10% bias and ~ 5% RSD for lipid classes
45 © 2015 AB Sciex
Conclusions
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
Three Key Elements of the Platform:
46 © 2015 AB Sciex
Acknowledgements
Sciex
• Baljit Ubhi
• Paul Baker
• Eva Duchoslav
• Larry Campbell
• Leo Wang
• Pauline Vollmerhaus
• Aaron Hudson
• Mark Cafazzo
• Marjorie Minkoff
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.
METABOLON
• Alex Conner
• Steve Watkins
• Annie Evans
• Richard Robinson
• Joseph Yu
• Luke Miller
• Sarada Tanikella
• Peter Zhu
• Corey DeHaven
Avanti Polar Lipids
• Walt Shaw
• Lisa Connelly