Where we’ve been, where we are, and where we’re going

Jeff McDonaldDepartment of Molecular GeneticsUT Southwestern Medical Center

Dallas, TXLIPID MAPS “Glue Grant” NIH U54 GM 69338

Lipid analysis by multiple "omic" mass spectrometry platforms

Lipidome of Human Plasma

588

Lipids in Human Serum

Total cholesterol ~ 2 mg/mL

Trace oxysterols < 2 ng/mL

> 6 orders ofmagnitude

Triglycerides ~ 1 mg/mLPhospholipids ~ 2 mg/mLSphingolipids ~ 0.5 mg/mL

Cholesteryl ester

pg/mL mg/mLWikipedia : Reference ranges for blood tests - by mass.svg

Cholesterol

mg/mL

2) Base hydrolysis

Organic-soluble(lipids)

Insoluble material/proteins

200μL serum + standards

1) Solvent extraction (DCM/MeOH)

General Steps of Lipid Extraction and Purification

Aqueous (MeOH/H2O)

Organic (lipids) (DCM/MeOH)

3) Liquid-Liquid Extraction4) Solid phase extraction

KOH, 60°C

Condition With hexane

Load samplein hexane

Hexanerinse

Non polarlipids

Sterols +oxysterols

23:1CHCl3:MeOH

5) LC-MS

Mass Spectrometry

• Triple Quadrupole (MRM)• Neutral Loss Scan• Precursor Ion Scan

HPLC

• Normal Phase• Reverse Phase• HELIC

Analytical Separation and Detection of Lipids

Mass Spectrometry

• Electron Ionization• Electron-Capture Negative

Ionizationo Methane

GC

• DB5-MS 40m 0.18mm i.d.o H2 carrier gas

• DB5-MS 30m 0.25o He Carrier gas

Sterols/Oxysterol4 Samples4 Extractions2 LC-MS Analysis2 GC-MS Analysis

Prenols/Cardiolipins3 Samples1 Extraction3 LC-MS Analysis

Neutral Lipids3 Samples2 Extractions1 LC, 3 MS Analysis

Phospholipids2 Samples2 Extractions2 LC-MS Analysis

Human Plasma

Lipidome

Fatty Acids/Eicosanoids2 Samples2 Extractions1 LC-MS Analysis1 GC-MS Analysis

Sphingolipids1 Sample3 Extractions5 LC-MS Analysis

Summary15 Samples14 Extractions14 LC-MS methods3 GC-MS methods100’s standards7 Expert Centers$ Millions/many hours

Measurement of the Plasma Lipidome

The Workhorses of Lipid Analysis

AB Sciex Triple QuadrupoleMass Spectrometers Agilent GC-MS

Analytical Standards Generated by Avanti Polar Lipids

• >650 analytical lipid standards–300 new lipid standards–Primary, deuterated, odd-chain

• Purity and stability–Packaging, storage, shelf-life– In-house analytical expertise

Eicosanoids and Fatty Acids from Cayman Chemical

100+ eicosanoid

MS-Based Methods Developed by LM Cores

lipidmaps.org

A Ten-Year Timeline of Lipidomics

2003 2013

LIPID MAPS pioneers and advances speciated, quantitative LC/GC-MS methods for lipids

“Shotgun” Lipidomics introduced and implemented by Hann and Gross

Broad-scale, targeted and untargeted LC-MS “Omics” approaches flourishSuizdak, Fiehn, Sumner, Newgard, Roddy, etc

Advanced, infusion-based lipidomic methods introduced

Sterols

Sphingolipids

Phospholipids

Neutral Lipids

Prenols

Eicosanoids/Fatty Acids

One Sample, Many Choices

One Sample, Many Choices

“Ursus Analyticus” – Thanks Avanti!

Infusion-Based MS Platform for Lipidomic Analysis

Small moleculemetabolome

Insoluble material/proteins

20uL human plasma

1) Sample Extraction(CH2Cl2/MeOH)

LEAP Robotic Infusion System AB SCIEX TripleTOF 5600+ MS with SelexION

Workflow for Infusion-Based Global Lipidomics

B/D Extractionof plasma

Infuse into MS

Acquire TOF Spectrum

Acquire MS/MSALL

SpectrumData

Analysis

1 2 3 4 5

Workflow for Infusion-Based Global Lipidomics

B/D Extractionof plasma

Infuse into MS

Acquire TOF Spectrum

Acquire MS/MSALL

SpectrumData

Analysis

1 2 3 4 5

Plasma+StdsAqueous

Organic (lipids)

Workflow for Infusion-Based Global Lipidomics

B/D Extractionof plasma

Infuse into MS

Acquire TOF Spectrum

Acquire MS/MSALL

SpectrumData

Analysis

1 2 3 4 5

Workflow for Infusion-Based Global Lipidomics

B/D Extractionof plasma

Infuse into MS

Acquire TOF Spectrum

Acquire MS/MSALL

SpectrumData

Analysis

1 2 3 4 5

TOF SpectrumScan 200-1200 Da30-40K resolution< 2ppm mass accuracy

Cholesterol

CE

PC

TGPE

DG

SM

Workflow for Infusion-Based Global Lipidomics

B/D Extractionof plasma

Infuse into MS

Acquire TOF Spectrum

Acquire MS/MSALL

SpectrumData

Analysis

1 2 3 4 5

m/z 666CE 18:2

Workflow for Infusion-Based Global Lipidomics

B/D Extractionof plasma

Infuse into MS

Acquire TOF Spectrum

Acquire MS/MSALL

SpectrumData

Analysis

1 2 3 4 5

m/z 666CE 18:2Product-ion Spectrum

m/z 666m/z 369

m/z 666

Neutral Loss 379 = FA 18:2

Workflow for Infusion-Based Global Lipidomics

B/D Extractionof plasma

Infuse into MS

Acquire TOF Spectrum

Acquire MS/MSALL

SpectrumData

Analysis

1 2 3 4 5

Hour Time Scale

30 minutes 5 minutes

Mining the Dataset Post-Acquisition

Lipids that contain a 18:1 fatty acid (NL 281Da)

MS/MSALL identifies 800+ MS features

Lipids that contain a PE head group(NL 141 Da)

Lyso Phosphatidylethanolamine

Cholesteryl ester C18:1Triacylglycerol with FA 18:1

Lipids that contain a PC head group(Prec 184 Da) Lyso Phosphatidylcholines

Phosphatidylcholine

Phosphatidylethanolamine

PCA Analysis – MS/MSALL Data

Liver

Plasma

Blank

MarkerView (AB Sciex)

Can Infusion-Based Metabolomics be Quantitative?

LPC 18:0

C 36:2 DAG

TAG 54:3

The Future of Lipidomics

• Chromatograhic-based quantitative analysis– HPLC resolving power and efficiency– MS sensitivity– MS acquisition speed– Flexible, parallel acquisition

• Infusion-based analysis– Resolution, mass accuracy, linearity– Data analysis software– Off-line HPLC followed by infusion-based MS

• Ion-mobility• Accessibility to standards

– Class-specific mixtures (primary and internal)

Acknolwedgements

• LIPID MAPS community• Phil Sanders – Eli Lilly• Brigitte Simons – AB Sciex

– Jennifer Krone– Mark Cafazzo– Lyle Burton

• AB-Sciex Academic Partnership Program