ANSWERS FOR SCIENCE. KNOWLEDGE FOR LIFE.

Baljit Ubhi, Ph.DASMS Baltimore, June 2014

SelexION™ Technology for Lipid Analysis: Pushing the Boundaries of Lipidomics

2 © 2014 AB SCIEX

Lipidomics Profiling Needs and Deliverables

Liver metabolismStåhlman, M. et al J Chromatogr B Analyt Technol Biomed Life Sci. 2009

3 © 2014 AB SCIEX

Fatty Acids Steroids LipidVitamins

Terpenes

Eicosanoids/Oxidized FA

Glycerophospholipids

Mono-, Di- and Triglycerides

Waxes Sphingolipids

Ether Phospholipids

Platelet-Activating

FactorPlasmalogens

Diacyl-Linked Phospholipids

CeramidesSphingomyelins

CerebrosidesGangliosides

Lipids play an essential role in human physiology:

• Metabolic homeostasis

• Cell signaling• Cell and organelle

structure

And disease:

• Inflammation• Cancer• Cardiovascular

disease• Diabetes• Inflammatory

bowel disease• Neurological

diseasesHalogenated Lipids

Oxidized Phospholipids

The LipidomeComprised of Multiple, Distinct Structural Lipid Classes

4 © 2014 AB SCIEX

PG

550 600 650 700 750 800 850 900 950

Mass/Charge (Da)

PCPEPA

PSPI

• Lipidomicspectra are incredibly complex

• MS/MS spectra generated on precursors in zones of isobaric overlap will contain product ions from other isobaric species

Isobaric Overlap of PhospholipidsExperiment: EMS scan of Bovine Heart Extract (BHE)

5 © 2014 AB SCIEX

1. Orifice Plate 2. Ion Mobility Cell 3. Curtain Plate

SelexION™ Technology Ion Mobility Device Components

• Robust, easy-to-install, hardware components:‒ No tools required‒ No cables‒ No need to break vacuum‒ Installation in about 2 minutes

6 © 2014 AB SCIEX

Separation waveform (SV): radially displaces ions towards one or the other electrode, depending upon high and low mobility characteristics

Compensation voltage (COV): restores the trajectory for a given ion or range of ions to allow them to transmit through the DMS deviceand enter the mass spectrometer

SV COV

ToMS

Gas/Modifierflow

Differential Mobility Spectrometry (DMS) is the term used for planar geometry

How Does DMS Separate Ions?

7 © 2014 AB SCIEX

SelexIONTM Technology Separates Phospholipid Classes

PC

PE

PA

PGPS

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

Proof of concept: DMS separates simple lipid mixtures

Implications: “Cleaner” quantitative data using mrm scan modes (esp., MRM HR); Improved qualitative analysis

Experiment: MRM Scan of 6 Phospholipid Standards with COV Ramp

8 © 2014 AB SCIEX

Effects of DMS on Complex Lipid AnalysisCan DMS help resolve lipids from a complex, biological lipid mixture?

Experiment 1: EMS Scan of Bovine Heart Extract; No DMS

450 500 550 600 650 700 750 800 850 900 950

Mass/Charge (Da)

1e6

2e6

3e6

4e6

5e6

6e6

7e6723.70

529.48

766.42 861.36 885.33747.41

786.39698.46794.38592.54480.32

-18 -16 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18COV (V)

8.3-1.5

2.7

5.80.7

-5.2

Experiment 2: EMS Scan of Bovine Heart Extract; DMS with COV Ramp

9 © 2014 AB SCIEX

An individual phospholipid sub-class can be extracted from an EMS scan based on its differential COV

• Simplifies LipidView™

Software searching

• Increases confidence in lipid identification

EMS TIC

Resolution of Phospholipid Sub-Classes in a Biological Lipid Extract by DMS

Experiment: EMS scan of bovine heart extract with ramped COV

10 © 2014 AB SCIEX

Relationship Between Dipole Moment and COV

-8

-6

-4

-2

0

2

4

6

8

3 3.5 4 4.5 5 5.5 6

CV

(V)

Model headgroup dipole moment (D)

PC 16:0_18:1 [-H +Cl-]

PE 16:0_18:1 [-H-]

PA 16:0_18:1 [-H-]

PS 16:0_18:1 [-H-]

PG 16:0_18:1 [-H-]

PI 16:0_16:0 [-H-]

Isopropanol as a chemical modifier in DMS

11 © 2014 AB SCIEX

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

12 © 2014 AB SCIEX

TOF MS Analysis of Lung Lipid Extract Using DMS

Experiment: TOF MS with COV ramp in negative ion mode;

Infusion of 100 μg/mL lung lipid extract

Fatty Acids

Phospholipids; Cardiolipin

13 © 2014 AB SCIEX

Fatty Acid Resolution by DMS

Fatty Acids1

1

2

2

3

3

Experiment: TOF MS with COV ramp in negative ion mode;

Infusion of 100 μg/mL lung lipid extract

16:118:2 18:1

16:0

18:0

Extracted Ion Chromatogram

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DMS Resolves Fatty Acid Cis/Trans Isomers

18:1 (cis)

18:1 (trans)

16:1 (cis)

16:1 (trans)

Resolution of cis/trans isomers usually requires chromatographic separation prior to analysis; DMS can remove this need, increasing the speed of FA analysis

Experiment: TOF MS with COV ramp in negative ion mode;

Infusion of 100 μg/mL lung lipid extract

15 © 2014 AB SCIEX

Resolution of Phosphatidylcholine (PC)

770.5307 792.5758 830.5917 854.5901

16 © 2014 AB SCIEX

Resolution of Phosphatidylethanolamine (PE)

PC O-30:0 + AcO is iso-elemental with PE 38:5

C43H77O7NP

XIC 750.5446

PC O-30:0 +AcO

PE 38.5

0.4 ppm for bothmolecules

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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 Range 1V

COV Range 0.1 V

COV for TAG

20 © 2014 AB SCIEX

SLICE: Structural Lipidomics Investigations using Chemical Effects

DMS-Separated Precursors

MS/MS ofSelected Precursors

DMS SeparationIn CV Space

DMS SeparationIn CV Space

• Molecules are separated in CV space prior to entering the MS

• Chemical modifiers enhance compound resolution

• Minimizes isobaric overlap

21 © 2014 AB SCIEX

Confident Identification of Isobaric Compounds

0.2u

Experiment: DMS MS/MSALL (NEG) of bovine heart extract

PC p16:0_18:2 PE p16:0_22:5

PE p16:1_22:4

CL 80:11 (20:4, 20:5, 18:1)

PG 18:1_18:0*

* 1st isotope

Duchoslav E., Campbell, J. L., Baker, P.R.S., Patterson B.T., ASMS 2013, ThP 28-586

22 © 2014 AB SCIEX

DMS Separation Improves Coverage and Confidence in Lipid Species Annotation

Sample type Acquisition strategy Polarity # lipids*

Bovine Heart

TOF MS+ 135- 225

combined 266

DMS+ 248- 340

combined 466

E. Coli

TOF MS+ 164- 138

both 174

DMS+ 81- 167

combined 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

23 © 2014 AB SCIEX

SelexION™ Technology for Lipidomic Analysis

• 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

Effective lipid resolution without

a need for complicated

chromatographic strategies

24 © 2014 AB SCIEX

Acknowledgements

AB SCIEX

• Paul Baker• Larry Campbell• Michael Jarvis• Eva Duchoslav• Brigitte Simons• Christie Hunter• Baljit Ubhi

Zora Biosciences

•Kim Ekroos•Tuulia Sylvanne

UCSD

• Ed Dennis• Oswald Quehenberger• Paul Norris• Aaron Armando

Eli Lilly

• Phil Sanders

LipidMAPS.org

Wollongong University

• Todd Mitchell• Steven Blanksby• Simon Brown• Al Maccarone

Avanti Polar Lipids

25 © 2014 AB SCIEX

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.

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ANSWERS FOR SCIENCE. KNOWLEDGE FOR LIFE.ANSWERS FOR SCIENCE. KNOWLEDGE FOR LIFE.

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