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Lipid Analysis with Differential Mobility Spectrometry Fadi Abdi, Ph.D. March 2013

Public Health Action Plan to Integrate Mental Health Promotion and

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Page 1: Public Health Action Plan to Integrate Mental Health Promotion and

Lipid Analysis with Differential Mobility Spectrometry

Fadi Abdi, Ph.D. March 2013

Page 2: Public Health Action Plan to Integrate Mental Health Promotion and

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

Page 3: Public Health Action Plan to Integrate Mental Health Promotion and

3 © 2012 AB SCIEX

• 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

Page 4: Public Health Action Plan to Integrate Mental Health Promotion and

4 © 2012 AB SCIEX

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

Page 5: Public Health Action Plan to Integrate Mental Health Promotion and

5 © 2012 AB SCIEX

QTRAP® 6500 system Ion Path with Ion Mobility Technology

Page 6: Public Health Action Plan to Integrate Mental Health Promotion and

6 © 2012 AB SCIEX

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

Page 7: Public Health Action Plan to Integrate Mental Health Promotion and

7 © 2012 AB SCIEX

Phospholipid (PL) Sub-Classes

Differential

charge site

topography

makes

phospholipids

ideal

candidates for

differential

separation by

SelexION DMS

Page 8: Public Health Action Plan to Integrate Mental Health Promotion and

8 © 2012 AB SCIEX

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

Page 9: Public Health Action Plan to Integrate Mental Health Promotion and

9 © 2012 AB SCIEX

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

Page 10: Public Health Action Plan to Integrate Mental Health Promotion and

10 © 2012 AB SCIEX

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

Page 11: Public Health Action Plan to Integrate Mental Health Promotion and

11 © 2012 AB SCIEX

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

Page 12: Public Health Action Plan to Integrate Mental Health Promotion and

12 © 2012 AB SCIEX

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

Page 13: Public Health Action Plan to Integrate Mental Health Promotion and

13 © 2012 AB SCIEX

DMS Resolves PC and Sphingolipids

Precursor Ions +184

Phospholipids Sphingomyelins

Page 14: Public Health Action Plan to Integrate Mental Health Promotion and

14 © 2012 AB SCIEX

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

Page 15: Public Health Action Plan to Integrate Mental Health Promotion and

15 © 2012 AB SCIEX

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

Page 16: Public Health Action Plan to Integrate Mental Health Promotion and

16 © 2012 AB SCIEX

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’

Page 17: Public Health Action Plan to Integrate Mental Health Promotion and

17 © 2012 AB SCIEX

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

Page 18: Public Health Action Plan to Integrate Mental Health Promotion and

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.

Page 19: Public Health Action Plan to Integrate Mental Health Promotion and

Thank You

Page 20: Public Health Action Plan to Integrate Mental Health Promotion and

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