Agilent LC/MS Work-flows for Advancing Lipidomics Research · PDF fileOutline Agilent hardware...

Agilent LC/MS Work-flows for Advancing Lipidomics Research

Sumit Shah, B.Pharm., Ph.D.

LC/MS Applications Scientist,

Agilent Technologies, Inc.

Lipidomics

Agilent Metabolomics/Lipidomics Applications Team

Dr. Anne Blackwell

Dr. Daniel Cuthbertson

Dr. Mark Sartain

Dr. Sumit Shah

Dr. David Weil

Dr. Jim Lau

Petrochemicals

BioFuels

Consumer Nutrition

Life Sciences

Pharma

BioPharma

Diagnostics

Outline

Agilent hardware and software solutions for implementing

different lipidomics work-flows

Lipid profiling of tissue extracts: Reversed phase and

normal phase chromatography

Fish oil lipidomics: Supercritical fluid chromatography

Ion Mobility mass spectrometry for cerebroside analysis

Differential expression analysis of human serum lipidome

Lipidomics: The systematic study of the entire lipid profile of a cell/tissue/organ/organism

Eicosanoids

Acetyl CoA

Isopentenyl-PP

Sphingolipids Phospholipids

Glycerolipids

Sterols

Prenols

Saccharolipids

Fatty Acids

Functions

- Structure

- Storage

- Energy

- Signaling

Molecular Diversity

Yetukuri L et al. Molecular Biosystems. 2008. 4: 121-127

Known Lipids From LIPID MAPS

LIPID MAPS: largest public lipid-only database

37,566 unique lipid structures as of May 3, 2013

Structures of lipids in the database come from several sources:

LIPID MAPS Consortium's core laboratories and partners

Identified by Lipid Maps experiments

Biologically relevant lipids manually curated from LIPID BANK, LIPIDAT, Lipid Library, Cyberlipids, ChEBI

Computationally generated

Database content

Fatty acyls 5797

Glycerolipids 7538

Glycerophospholipids 8001

Sphingolipids 4318

Sterol lipids 2678

Prenol lipids 1200

Saccharolipids 1293

Polyketides 6741

Current Challenges in Lipidomics

A deep characterization of the lipid composition of a single plasma reference sample

required the combined efforts of six leading lipidomics laboratories.

Quehenberger O et al. Journal of Lipid Research. Nov. 2010 51(11) 3299-305

588 lipids identified

and quantified

Untargeted, Discovery based Data Mining Workflow A

gile

nt

LC

MS

and G

CM

S

Feature

Finding

MassHunter

Profinder (NEW)

MassHunter

Qual

MassHunter

Quant

Alignment &

Statistics

Statistics

Visualization

Identify

Annotation &

Identification

Pathway

Analysis

Pathway

Analysis

LCMS and GCMS Data can be analyzed

together in the same project

Mass Profiler Professional

Separate &

Detect

Compound Identification with Metlin®

Most Extensive Database and MS/MS Library for Metabolomics

METLIN, an accurate mass LC-MS/MS library (Q-TOF based)

Compounds with MS level information – 64092

Lipids with MS level information – 31011

Human metabolites with MS level information – 19714

Compounds with MS/MS spectral information – 8040 (448 lipids)

MS/MS of mono-isotopic molecular ion

MS/MS in positive and/or negative ionization mode

Fragmentation performed at three collision energies: 10, 20 and 40

MS/MS spectra are computer and manually reviewed for quality

Lipidomics Software Solution

SimLipid**

Comprehensive lipidome MS and MS/MS database/analysis

Lipids with MS level information – 36299

In silico MS/MS fragmentation information/matching

SimLipid 4.1 supports four other MS vendors’ data but Agilent data is the only one

that:

Allows import/searching of compounds (MassHunter Qual cef files) rather than raw

spectra

Does MS/MS pattern matching of multiple adducts (e.g. ammonium, sodium)

Can annotate the cef files with the identified lipids for further processing

Has a complete workflow with MassHunter Qual, MPP, and Pathway Architect for

biological contextualization

Lipidomic Profiling of Tissue Extracts: Impact of Chromatography

Samples

Well-characterized total lipid extracts were

purchased from Avanti® Polar Lipids (Alabaster,

AL)

Dried lipid extracts were reconstituted in 2:1

chloroform/methanol and further diluted in Mobile

Phase A to a concentration of 200ng/µL

Injections of 5µL (1µg extract) were analyzed by

reversed- or normal-phase LC-MS

Agilent 1290 UHPLC with 6540 QTOF

Reversed-phase (RP) LC Method

Eclipse Plus RRHD C18 2.1x100mm, 1.8µm column @ 50°C

Mobile phase A: 0.1% CH3COOH, 5mM CH3COONH4 in

[CH3]2OH:CH3OH:H2O (5:1:4)

Mobile phase B: 0.1% CH3COOH, 5mM CH3COONH4 in

[CH3]2OH:H2O (99:1)

0.35mL/min flow-rate, 0% B for 3min, 20% B at 5min, 30% B

at 25min, 95% B at 35min

Agilent 1290 UHPLC with 6540 QTOF

Normal-phase (RP) LC Method

Zorbax Rx-Sil C18 2.1x100mm, 1.8µm column @ 25°C

Mobile phase A: 0.1% CH3COOH, 5mM CH3COONH4 in

[CH3]2OH:C6H14:H2O (58:40:2)

Mobile phase B: 0.1% CH3COOH, 5mM CH3COONH4 in

[CH3]2OH:C6H14:H2O (50:40:10)

0.3mL/min flow-rate, 0% B for 5min, 100% B at 34min

Lipid Profile of Cardiac and Brain Extracts (RP, +ESI)

Brain Extract

Overlaid 2,905 Extracted Compound Chromatograms

939 Annotated Lipids

Cardiac Extract

Overlaid 2,955 Extracted Compound Chromatograms

948 Annotated Lipids

Overlaid 75 Annotated Cer ECCs Ceramides

Lipid Profile of Liver Extract (RP, +ESI) Liver Extract

Overlaid 4052 Extracted Compound Chromatograms

1372 Annotated Lipids

Overlaid 184 Annotated TAG ECCs TAGs

Overlaid 282 Annotated DAG ECCs DAGs

Lipid Profile of Liver Extract (RP, +ESI)

Liver Extract

Overlaid 1679 Extracted Compound Chromatograms

528 Annotated Lipids

Lipid Profile of Liver Extract (NP, +ESI)

Lipid Profile of Liver Extract (NP, +ESI) Separation by Lipid Class

Ceramides

Sphingosines PE

LPE

PI

PC

SM

LPC

17

Missing neutral lipids: DAGs, TAGs, Sterols

Summary: Comparison of LC Strategy

Reversed-phase LC-MS

Normal-phase LC-MS

Advantages

Many lipid classes resolved = comprehensive

Sensitive = most # molecular features

Retention times very reproducible = alignment made

easy

Advantages

RT behavior of lipid classes allows more confident

lipid annotations

User can quickly assess lipid class differences in

samples from chromatograms

Two alternative LC separation modes each provide selective advantages in lipidomics

analyses: the best choice may depend on the application

Choice of chromatographic method will impact the lipids that are resolved and annotated

Combining multiple chromatographic methods provide for deeper and comprehensive

mining of a lipidome

What is Supercritical Fluid Chromatography (SFC)?

A separation technique similar to HPLC in terms of

hardware, software and even columns

Form of normal phase chromatography, widely used for

thermally labile compounds and separation of chiral

compounds

Supercritical CO2 is the main component in the mobile

phase (polarity ~ n-heptane)

Co-solvents such as methanol, isopropanol and ethanol

added to modify the polarity of the system

In theory, SFC can be used for any molecule that is

soluble in methanol and/or any solvent of lesser polarity

Polar and non-polar lipids can be separated in one run

Polar Lipids (PE’s, PC’s) elute late in chromatogram

ESI spectra are consistent with LC ESI libraries

SFC w/ pure CO2

CO2 w/ organic modifiers

CO2 w/ modifiers + additives

CO2 + modifiers +

additives + water

Normal Phase HPLC

Reversed Phase HPLC

Ion Pairing

HILIC

Ion

Chromatogr.

Where Does SFC Fit Relative to HPLC?

Solute Families

0

200

400

600

800

1000

1200

1400

1600

M a

s s (

D a )

0 1 2 3 4 5 6 7 8

Retention Time (min)

750

900

Ma

ss

, D

a

2.2 2.4 Time (min)

The fish oil was purchased as a commercial dietary supplement in gel capsule and diluted either

1:100 or 1:1000 with isopropyl alcohol.

Fish Oil Lipid Analysis with SFC/MS

Agilent 1260 SFC with 6540 QTOF

Zorbax SB300 C18 4.6x150mm, 3.5µm column @ 60°C

Mobile phase A: SFC CO2

Mobile phase B: 5mM CH3COONH4 in CH3OH

3mL/min flow-rate, 3% B for 5.5min, 60% B at 11min

≈ 10:1 split into QTOF with CH3OH make-up

~ 2000 features

~ 400 annotated lipids

DGs Vit D precursors

2beta-methyl-1beta,25 dihydroxycholecalciferol

TGs

Extracted Ion Chromatograms of Fish Oil SFC/MS

TGs: C53H98O6, C53H96O6, C53H94O4

C53H98O6

C53H96O6

C53H94O6

23

Summary: Practical Advantages of SFC

Orthogonal to reversed phase HPLC

Amenable to UHPLC method development

Sensitivity, dynamic range, and capacity like HPLC

Low solvent consumption/low waste generation

Lower operating cost

Great for separation of isomers in complex lipid samples

Green!

Agilent 6560 Ion Mobility-QTOF

Better IM resolution

Higher IM sensitivity

Resolve complex samples

Direct measurement

of Ω

Preserve molecular structures

front funnel trapping funnel drift cell rear funnel

tdrift

Detector

Analyte

Ions

Gating

Optics

Ion Mobility Cell

t0

VH VL

Basic Operational Principle of Ion Mobility

For Conventional DC Uniform Field IMS

Electric Field

Stacked ring ion guide gives linear field

Direct determination of accurate Ω 𝑣 = 𝐾 𝐸 ∝𝑒 𝐸

𝑃 𝑇 Ω

Tetraalkylammonium Salts

+2 ions

+3 ions

+1 ions

+4 ions

Ion

Mo

bilit

y D

rift

Tim

e (

ms)

Mass (Da)

0

0

20

40

50

500 1000 1500 2000

10

30

60

70

L-α-phosphotidylethanolamines (PE)

TAA-3

TAA-16

TAA-12

TAA-10

TAA-8 TAA-7

TAA-6

TAA-5

TAA-4

Lipid Analysis with 6560 IM-QTOF

Ion

Mo

bilit

y D

rift

Tim

e (

ms)

Mass (Da)

0

0

20

40

50

500 1000 1500 2000

10

30

60

70

PE 60:N PE 62:N

PE 64:N

PE 33:N PE 35:N

PE 37:N PE 39:N

PE 41:N

PE 23:N PE 21:N

PE 19:N

PE oligomers (+1)

PE oligomers (+2)

L-α-phosphotidylethanolamines (PE)

Lipid Analysis with 6560 IM-QTOF

Lipid Analysis with 6560 IM-QTOF Io

n M

ob

ilit

y D

rift

Tim

e (

ms)

Mass (Da)

0

0

20

40

50

500 1000 1500 2000

10

30

60

70

PE 35:(6-2) PE 37:(8-4) PE 39:(10-6) PE 33:(4-2)

+Na +K

Mass (Da)

740 760 770 780 790 750 800 810 820

PE 60:N PE 62:N

PE 64:N

PE 33:N PE 35:N

PE 37:N PE 39:N

PE 41:N

PE 23:N PE 21:N

PE 19:N

PE oligomers (+1)

PE oligomers (+2)

Summary for Ion Mobility-QTOF

Added dimension of separation based on size, charge and

ion structure

Resolve and characterize complex samples using

LC/IM/MS analysis while maintaining high sensitivity

Direct method to calculate accurate collision cross

sections for added analytical confirmation

Means to study structural diversity of target molecule

Female Serum, +ESI

Male Serum, +ESI

Female Serum, -ESI

Male Serum, -ESI

Agilent 1290 UHPLC with 6550 QTOF

Eclipse Plus RRHD C18 2.1x100mm, 1.8µm column @ 60°C

Mobile phase A: 0.1% CH3COOH, 1mM CH3COONH4 in H2O

Mobile phase B: 0.1% CH3COOH, 1mM CH3COONH4 in

[CH3]2OH:CH3OH (70:30)

0.3mL/min flow-rate, 20% B for 2min, 100% B at 22min

Human serum was collected and pooled from

healthy (males and females) subjects

(Innovative Research Inc.)

Lipids were extracted from 100µL (0.22µm

filtered) serum with 700µL ice-cold 1:1

[CH3]2OH:CH3OH containing 1% CH3COOH

Inject 10µL of lipid extract for LC/MS analysis

Lipidomics Profiling of Human Serum by LC/MS

Human serum equivalent to 1.25µL was used for the lipidomics studies

Feature Finding using ProFinder Batch Analysis (+ESI)

Female Serum

Male Serum

Male Serum

Female Serum

Statistical Analysis in Mass Profiler Professional (MPP)

Filtered 9083 features that are present in all three replicates (+ESI) in any one group with CV<15%

Differential Expression Analysis in MPP (+ESI)

Cluster Analysis in MPP (+ESI)

Female Serum Male Serum

Select List of Statistically Significant Annotated Lipids +ESI

-ESI

LC/MS/MS Identification of 1,2-dipalmitoyl-sn-glycero-3-PC

??

LC/MS/MS Identification of 1,2-dipalmitoyl-sn-glycero-3-PC

1,2-dipalmitoyl-sn-glycero-3-PC

MS/MS @ 40

Metlin Spectral Library

1,2-dipalmitoyl-sn-glycero-3-PC

C18 LC/MS +ESI

Female Serum

Male Serum

Summary of Human Serum Lipid Profiling

Filter features present in all replicates in a group with CV<15%

Features searched against SimLipid® Database

9083 features identified in +ESI mode (1470 annotated lipids)

4423 features identified in –ESI mode (790 annotated lipids)

222 annotated lipids common between +ESI and –ESI modes

A combined total of 2038 lipids were annotated in human serum samples

-ESI

568

+ESI

1248 222

Summary

Highlighted Agilent hardware and software solutions to

conduct lipidomics research

Demonstrated most commonly used lipidomics work-flows

Presented the advantages of SFC as an additional

separation technique for lipid analysis

Introduced the application of ion mobility mass spectrometry

for complex lipid analysis