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