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

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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

9 © 2015 AB Sciex

Simplifying the Complexity

Powered by METABOLON®

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!

How the LipidyzerTM Platform Works

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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:

23 © 2015 AB Sciex

Lipidomics Workflow Manager: Dashboard

Simple and Guided Workflow

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Lipidomics Workflow Manager: Dashboard

System Suitability Tests and Automated Tuning for SelexION

25 © 2015 AB Sciex

Lipidomics Workflow Manager: Sample Login and Metadata Entry

Flexible Import

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

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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

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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

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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

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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

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