SelexION™ Technology for Lipid Analysis: Pushing the ... · ANSWERS FOR SCIENCE. KNOWLEDGE FOR...
Transcript of SelexION™ Technology for Lipid Analysis: Pushing the ... · ANSWERS FOR SCIENCE. KNOWLEDGE FOR...
ANSWERS FOR SCIENCE. KNOWLEDGE FOR LIFE.
Baljit Ubhi, Ph.DASMS Baltimore, June 2014
SelexION™ Technology for Lipid Analysis: Pushing the Boundaries of Lipidomics
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Lipidomics Profiling Needs and Deliverables
Liver metabolismStåhlman, M. et al J Chromatogr B Analyt Technol Biomed Life Sci. 2009
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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
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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)
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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
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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?
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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
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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
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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
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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
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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
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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
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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
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Resolution of Phosphatidylcholine (PC)
770.5307 792.5758 830.5917 854.5901
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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
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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
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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
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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
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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
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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
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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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