separating lipid isomers with lc -ims-ms measurements to
TRANSCRIPT
Separating Lipid Isomers with LC-IMS-MS Measurements
to Understand Their Role in Biochemical Processes
Erin S. BakerKristin E. Burnum-Johnson, Jennifer E. Kyle, Xing Zhang, Matthew E. Monroe, Yehia M. Ibrahim, Thomas O. Metz and Richard D. Smith
Pacific Northwest National Laboratory
E
in out
Pulse of 2 ions with same m/z but different shape
Different conformers separate in time with peak heights representing the amount of each
Drift Cell
Drift Time
• Lipids are composed of the same simple double bond and fatty acid components, resulting in numerous isomers
• IMS is a very fast separation technique which provides structural information
• In conjunction with LC, IMS-MS provides the capability to resolve many more isomers and reduce false discovery rates
Why use IMS in lipidomic studies?
Glycerophospholipids
Fatty Acids
Glycerolipids
Sterols
Sphingolipids
6
Eicosanoids
Importance of fatty acids
Fatty acids are key components of lipids, giving them their hydrophobic properties
Quehenberger O, et al. Lipidomics reveals a remarkable diversity of lipids in human plasma. Journal of lipid research. 2010;51(11):3299-305.
Fatty acid analysis with IMS
cis 9 cis 6
16:1
cis 9cis 6
16:1
cis 9
18:1
cis 6 cis 11
cis 9 trans 9 or 11 22:1
22:0cis 1318:0
cis 11
cis 9
trans 11
trans 9
18:0
18:1
Fatty acid analysis with IMS
Lowest energy structures from AMBER molecular dynamics simulations
Lyso-PL
TG
DG
Cer
SM
Diacyl-PL
DGDG
MGDG
Both LC and IMS are able to separate different lipids types, but each gave distinct separations
Lipid database
LC IMS
Paglia G, et al. Applications of ion-mobility mass spectrometry for lipid analysis. Analytical and bioanalytical chemistry. 2015.
May JC, et al. Conformational ordering of biomolecules in the gas phase: nitrogen collision cross sections measured on a prototype high resolution drift tube ion mobility-mass spectrometer. Anal Chem. 2014;86(4):2107-16.
Phospholipid separations with IMS
CompactnessPE < PE(P) = PS = PG < PC
Shvartsburg AA, et al. Separation and classification of lipids using differential ion mobility spectrometry. J Am Soc Mass Spectrom. 2011;22(7):1146-55.
LC IMS
IMS was able to further separate the lipid subgroups
cis/trans double bond analysis
PE(18:1/18:1) (cis-Δ9)
PE(18:1/18:1) (trans-Δ9)
PE(18:0/18:0)
PE(18:1/18:1) (cis-Δ9) PE(18:1/18:1) (trans-Δ9)
sn-1/sn-2 positional analysis
Size Comparison (Relative cross sections)
PC(14:0/16:0) < PC(16:0/14:0)
PC(16:0/18:0) < PC(18:0/16:0)
PC(18:1/16:0) < PC(16:0/18:1)
PC(14:0/16:0)
PC(16:0/14:0)
sn-2
sn-1 sn-3
1
Elution Time (min)
Nor
mal
ized
Inte
nsity
Biological sample complexity
Peaks shown for 1-s acquisition from m/z range 682-882
90-min LC run for mouse tissue sample (>400 identifications)
1
Elution Time (min)
Nor
mal
ized
Inte
nsity
PossibilitiesPE(P-16:1/18:0)PE(P-18:0/16:1)PE(P-16:0/18:1)PE(P-18:1/16:0)
Biological sample complexity 2 Co-eluting Lipids (0.036 Da difference)
PC(P-18:0/20:4)
PE(18:1/20:4)
90-min LC run for mouse tissue sample (>400 identifications)
3 Co-eluting Isomers (same mass)
LysoPCs as disease biomarkers
sn-2PC(0:0/16:0)
sn-1PC(16:0/0:0)
sn-1
sn-1
sn-2
sn-2
Different lysoPCs have been linked to diseases so we started looking at lysoPC16:0 in different samples
PC16:1 in Huh7.5.1 Human Hepatoma Cell Line
sn-2
sn-1
sn-2
sn-1
Cis forms are dominant, trans forms are <10% & immediately follow in elution time (denoted with )
sn-2sn-1
*
*
*
Collaboration with Steve Polyak at University of Washingtonon silymarin-treated Huh 7.5.1 cells
LC isomer analyses in biological samplesPC16:0 in Human Embryonic Epithelial Kidney Cell Linem/z = 496.3403
PC16:1 in Human Embryonic Epithelial Kidney Cell Linem/z = 494.3246
24 24
Drif
t Tim
e (m
s)
Drif
t Tim
e (m
s)
32
28
32
28
sn-2
sn-1
PC16:1 in Huh7.5.1 Human Hepatoma Cell Linem/z = 494.3246
PC16:0 in Human Embryonic Epithelial Kidney Cell Linem/z = 496.3403
sn-2sn-1
LC-IMS-MS isomer analyses in biological samples
sn-2
sn-1
1. Observed separation of sn-1 and sn-2 lysoPCs by LC and IMS
2. Two different double bond positions (P1 and P2) in the Hepatoma Cell Line
3. From IMS size, we hypothesize P1 is in the middle and P2 is near the head group (since it is larger)
4. Cis forms are dominant, trans forms are <10% and immediately follow in elution time (denoted with )
24 24
Drif
t Tim
e (m
s)
Drif
t Tim
e (m
s)
32
28
32
28
sn-2(P1)
sn-1 (P1)sn-2
(P2)
sn-1(P2)
LC-IMS-MS isomer analyses in biological samplesPC16:1 in Huh7.5.1 Human Hepatoma Cell Linem/z = 494.3246
PC16:0 in Human Embryonic Epithelial Kidney Cell Linem/z = 496.3403
sn-2
sn-1
sn-2(P1)
sn-1 (P1)sn-2
(P2)
sn-1(P2)
PC16:1 in Huh7.5.1 Human Hepatoma Cell Line 24-hr Treatmentsn-1 (P1)
sn-1(P2)
sn-2(P1)
24 24
Drif
t Tim
e (m
s)
Drif
t Tim
e (m
s)
32
28
32
28
sn-2(P2)
LC-IMS-MS isomer analyses in biological samplesPC16:1 in Huh7.5.1 Human Hepatoma Cell Linem/z = 494.3246
PC16:0 in Human Embryonic Epithelial Kidney Cell Linem/z = 496.3403
PC 34:1(16:0/18:1)
PE 34:1(16:0/18:1)
PC 34:2(16:0/18:2)
PI 34:2(16:0/18:2)
PC 40:6(18:0/22:6)
PI 40:6(18:0/22:6)
PC 36:4(16:0/20:4)
PI 36:4(16:0/20:4)
PC 38:4(18:0/20:4)
PI 38:4(18:0/20:4)
PE 38:5(18:1/20:4)
PI 38:5(18:1/20:4)
MALDI imaging and IMS lipid quantification
Good correlation with phospholipids observed in MALDI imagingMALDI imaging only identified ~30 lipids, LC-IMS-MS identified ~400 lipids
M Pole
AM Pole
Top
BottomEmbryo
PC 36:4(16:0/20:4)
PI 36:4(16:0/20:4)
PC 38:4(18:0/20:4)
PI 38:4(18:0/20:4)
PE 38:5(18:1/20:4)
PI 38:5(18:1/20:4)
IMS lipid isomer analysis
PE(20:4/18:1)
PE(18:1/20:4)
PI(20:4/18:1)
PI(18:1/20:4)
Summary
LC-IMS-MS:
• Separates fatty acid and complex lipid isomers
• Use of 3-dimensional LC-IMS-MS information increases the confidence of lipid identifications
• Allows quantification of potentially important isomeric changes in disease states
Acknowledgements• National Institute of Environmental Health
Sciences of the NIH (R01ES022190)• PNNL Laboratory Directed Research and
Development Program• Environmental Molecular Sciences
Laboratory
Biological Separations & Mass Spectrometry Group