Omics Applications
SelexION® Differential Mobility Spectrometry Technology
RUO-MKT-11-6677-A
© 2017 AB Sciex
• Differential Mobility Separation (DMS)
• Installation / removal of DMA in < 2mins – no tools
required
A New Dimension in Selectivity
SelexION® Technology
DMS
cell Curtain
Plate Source
Extension
Ring
© 2017 AB Sciex
SV COV
Gas
flow To
MS
• Planar geometry
• Gas flow towards MS draws ions
(transport gas)
• Asymmetric waveform applied
which alternates between high field,
K(E) and low field, K(0) – separation
voltage (SV)
‒ Moves charged ion back and
forth between plates
‒ Ion will have net drift base on its
high and low field mobility
• Compensation voltage (COV) is
small DC offset between the plates
– filtering voltage
Differential Mobility Seperation - DMS
Waveform
DMS Dimensions
1x10x30 mm
© 2017 AB Sciex
• Polar modifiers can be added into
transport gas (curtain gas)
• Enhances the formation of clusters in
field-dependent way, which amplifies
the high and low field mobility
differences
‒ Dynamic cluster –decluster model
• Uniqueness of these cluster
interactions
‒ Increase selectivity of the
separation
‒ Decrease low field mobility relative
to high field
‒ Which increases the compensation
voltage (CV) and therefore the
peak capacity
Modified Transport Gases Improve Separations
© 2017 AB Sciex
Lipidomics Applications
© 2017 AB Sciex
Experiment: EMS (-) scan of Bovine Heart Extract (BHE)
PG
550 600 650 700 750 800 850 900 950
Mass/Charge (Da)
PC
PE
PA
PS PI
• Lipidomic spectra are very
complex
• Problem: The Q1 isolation
window during MS/MS is
~1.2 Da, which increases
the number of potential
isobars.
• MS/MS spectra generated
on precursors in zones of
isobaric overlap will contain
product ions from other
isobaric species
Isobaric Overlap of Phospholipids
Challenges in Lipidomics Analysis Click
for
more
info
© 2017 AB Sciex
• Possible to achieve baseline
separation of all six
phospholipids without using
chromatography.
• As a proof of concept, the
phospholipid classes are
separable by DMS, which
implies elimination of inter-
class isobaric interference
and improved qualitative and
quantitative lipidomic data.
Separation of Phospholipid Classes
Lipidomics Applications
PC
PE
PA
PG
PS
PI
Experiment: MRM (-) scan of 6 phospholipid standards with COV ramp
Click
for
more
info
© 2017 AB Sciex
• Isolating individual glycoforms of
cerebrosides, such as Galβ1-1'Cer and
Glcβ1-1'Cer, difficult to achieve due to the
virtually identical structures of these
isobaric lipids, whose only difference being
the stereochemistry of the 3’-
hydroxylgroup.
• Differential Mobility Separation (DMS)
effectively resolves these two isomers to
enable independent confirmation and
quantitation.
Separation of Glycosylceramides (Cerebrosides)
Lipidomics Applications
Galactosylceramide and glucosylceramide
standards were infused individually (top) or
together (bottom) and analyzed using SelexION®
Technology.
Click
for
more
info
© 2017 AB Sciex
• Ether-linked and diacyl-linked
phospholipids are near isobars
• Necessity to resolve ether-linked and
diacyl-linked species to characterize
their molecular species compositions
• Even with high resolution MS, product
ion analysis depends on a low-resolution
isolation step that would generate a non-
specific, convoluted MS/MS spectrum
from the nearly isobaric compounds
Resolution of Ether- and Diacyl-Linked Phospholipids
Lipidomics Applications
DMS allows resolution of these phospholipid sub-
classes.
Click
for
more
info
© 2017 AB Sciex
• Ability to distinguish these iso-elemental species is critical for the
characterization, identification and quantitation of lipids
• Using SelexION® Technology on a TripleTOF® 5600 System, a lung lipid
extract was infused and the DMS CoV was ramped from -40 to 20V in the
negative ion mode.
Separation of Iso-Elemental Lipid Species
Lipidomics Applications
An XIC of the
750.5446 m/z peak
reveals two peaks in
the corresponding
ionogram Isopropanol
used as DMS modifier
Click
for
more
info
© 2017 AB Sciex
• Lipid molecular species can have the
same MRM transition, which leads to
interfering peaks in the LC-MRM
chromatogram
• This can cause significant problems in
quantitation.
• An MRM transition for the molecular
species PE 40:5 yielded an LC
chromatogram with significant isobaric
interference (top).
• The target peak is actually a shoulder
on an adjacent peak, which prevents
peak integration and accurate
quantitation..
Quantitative Lipid Analysis
Lipidomics Applications
Pooled human serum lipid extract was analyzed a QTRAP®
5500 LC-MS/MS System equipped with SelexION®
Technology for targeted lipid quantitation
Click
for
more
info
© 2017 AB Sciex
• Sphingomyelins (SM) share a common,
diagnostic fragment with
phosphatidylcholines (PC) - the
phosphocholine head group (m/z 184,
positive ion mode).
• High resolution mass spectrometry can
resolve these two classes of lipids
• SM and PC are resolved from one
another using lipid class-specific
compensation voltages (CoV) prior to
MS analysis that removes isobaric
interference and generates class-
homogenous ion spectra.
Resolution of Sphingomyelins
Lipidomics Applications Click
for
more
info
© 2017 AB Sciex
• The Lipidyzer Platform is equipped with SelexION® technology which allows
the separation of phospholipids by their head groups before subsequent MRM
analysis.
Separation of Phospholipids
Lipidomics Applications
-18 -16 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8
COV (V)
0%
20%
40%
60%
80%
100%
LPC LPE PEPC SM
Click
for
more
info
© 2017 AB Sciex
• Top: LC-MS/MS analysis of an ethanol
extract from peritoneal cells monitoring
the SRM transition m/z 335→19519. DMS
voltages turned OFF
• Middle and bottom: Analysis of murine
peritoneal cell ethanol extracts using LC--
MS/MS with DMS voltages turned ON.
• SelexION® Technology allows the
resolution of leukotrienes in complex
samples
Separation of Leukotrienes
Lipidomics Applications Click
for
more
info
© 2015 Analytical Chemistry
© 2017 AB Sciex
• Another class of biologically relevant isomeric substances we investigated using
the SelexION® Technology for was the separation of PD1 and PDX. Direct
infusion data for the successful separation of these two isomers is highlighted
below.
• A 50 ng/mL solution was used for DI experiments with low resolution gas settings
and a SV of 4500V.
Separation of Protectins
Lipidomics Applications Click
for
more
info
© 2015 Analytical Chemistry
© 2017 AB Sciex
Reduction of Background Interference to Enable Retinoic Acid Analysis
Lipidomics Applications
Common Method
LC-APCI with -MRM
Pooled Liver Extract PA1
13-cis-RA
9-cis-RA
at-RA
?? ??
at-RA
LC-DMS-ESI with -MRM
Pooled Liver Extract PA1
Isobaric overlap and high
background make
quantitation impossible
Confident and accurate
quantitation
© 2017 AB Sciex
Isolation of Individual Lipid Classes for Analysis with MS/MSALL
Lipidomics Applications
PC MS/MSALL – CoV = -6.5
• Clean MS/MS data without isobaric contamination
• Reduced background
• Improved ID confidence
© 2017 AB Sciex
Removal of Isobaric Interferences of Phospholipids
Lipidomics Applications
Experiment: TOFMS analysis of BHE with DMS; 3 separate experiments with PL class-specifc CoV values
© 2017 AB Sciex
Correct Identification of PC Lipid Molecular Species
Lipidomics Applications P
C
All
Pre
curs
ors
PC 16:0/22:6 (-CH3)
PC 16:1/22:5 (-CH3)
PC 16:0/16:1 (+AcO)
PC 16:1/18:2 (+Cl-)
PC 16:0/16:1 (+AcO)
PC 16:1/18:2 (+Cl-)
PC O-15:0/20:3 (+Cl-)
Experiment: Product ion spectrum (TOF MS/MS) of bovine heart extract (m/z 790.6); DMS tuned for PC
© 2017 AB Sciex
Metabolomics Applications
© 2017 AB Sciex
• The intermediates common to glycolysis and gluconeogenesis are among the
most difficult to resolve using mass spectrometry.
Isobaric Overlap
Challenges in Metabolomics Applications Click
for
more
info
© 2017 AB Sciex
• 3-Phosphoglycerate (3PG) and 2-
phosphoglycerate (2PG), two
isomers from the
glycolysis/gluconeogenesis
pathways, cannot be distinguished
based on their m/z.
• However, at increased separation
voltage (SV), each isomer has a
different compensation voltage
(COV) and thus can be separated.
• A similar separation can be
obtained for phosphoenolpyruvate
(PEP), another intermediate of the
glycolysis/gluconeogenesis
pathways.
Separation of Glycolysis Pathway Metabolites
Metabolomics Applications
3pg 2pg
SV 3625
3pg+2pg
SV 1750
3pg+2pg
SV 500
3pg 2pg
Inte
nsit
y
PEP
3PG
2PG
PEP
3PG
2PG
PEP
3PG+2PG
PEP+3PG+2PG SV = 1000
SV = 2000
SV = 2750
SV = 3000
Click
for
more
info
© 2017 AB Sciex
• DMS parameters were tuned by infusing pooled analyte solutions and ramping
separation voltage (SV) and compensation voltage (CoV).
• SelexION® technology enabled accurate quantitation with typical detection
levels of <2nmol on column.
Separation of Glycolysis Pathway Metabolites
Metabolomics Applications
© 2017 AB Sciex
• SelexION® technology enabled accurate quantitation with typical detection
levels of <2nmol on column.
Separation of TCA Cycle Metabolites
Metabolomics Applications
© 2017 AB Sciex
Separation of TCA Cycle Metabolites
Metabolomics Applications
Maleic acid Fumaric acid
Maleic acid Fumaric acid
DMS On
DMS Off
© 2017 AB Sciex
• Protection of flux isotope whilst removing background interference
Flux Analysis on TripleTOF® System applying SWATH® Acquisition
Metabolomics Applications
Without DMS With DMS
C12
C13
© 2017 AB Sciex
Proteomics Applications
© 2017 AB Sciex
• Single ion monitoring (SIM) of +6 and +7 charge state of
BNP with and without DMS.
• DMS Off – Significant interference is observed in blank
matrix for the 6+ charge state and background noise for
the 7+
• DMS On – Background / interference at elution time of
peptide significantly drops
• Signals from both charge states at 128 and 640 pg/mL
can now be seen above the noise
• Ion mobility separates the interfering matrix peaks away
from the BNP peptide of interest thus providing a better
S/N and a substantially improved LLOQ
High Selectivity of Peptides
Improving LLOQs of Peptides in Matrix Click
for
more
info
© 2017 AB Sciex
• Poor fragmentation of large peptides can
sometimes limit detection with MRM,
making SIM a more desirable approach,
although often limited in selectivity
• Top – MRM signal of 30 kDa peptide in
plasma at 125 ng/mL
• Middle – SIM signal of same peptide in
plasma at 16 ng/mL
• Bottom – DMS + SIM at 16 ng/mL
provides large improvement in S/N
• An LLOQ of 4 ng/mL for a 30 amino acid
peptide (~4 KDa) was achieved in protein
precipitated rat plasma, an ~30x
improvement over the MRM analysis
strategy
Separation and Quantitation of Large Peptides
Improving LLOQs of Peptides in Matrix Click
for
more
info
© 2017 AB Sciex
• Isobaric phosphopeptides can be separated by DMS
Separation of NpSTLpSEEDYIEK and NpSTLSEEDpYIEK
Localization - Di-Phosphorylated Peptides
y7
y5
y4
y4
y8-98 y3
y5
y6-18
y6
0.5 1.0 1.5 2.0
Time, min
10.0
12.5
15.0
0.5 1.0 1.5 2.0 0
2.0e5
1.03
Inte
nsity,
cp
s
Co
V, V
200 400 600 800
Mass/Charge, Da
0
4.5e4
632.2 858.2
747.2 380.24
389.3
987.2
NpSTLpSEEDYIEK
NpSTLSEEDpYIEK
200 400 600 800 0
2.5e5
552.4
994.3 389.4
796.4 667.4
778.4 925.4 129.1
y7-18
y4
Click
for
more
info
© 2017 AB Sciex
0.5 1.0 1.5
Time, min
13.00
14.25
15.5
16.75
18.00
19.25
Inte
nsity,
cp
s
• Acetylated peptides with different acetylated lysine residues can also be
successfully separated by SelexION® Technology
Separation of KAcQLATKAAR and KQLATKAcAAR
Localization - Variants of Acetylated Peptides
KQLATKAcAAR
KAcQLATKAAR
200 400 600 800 0
1.0e7
514.9
129.1
772.4
900.4
659.3 441.9 588.3 317.3
883.5 370.4
y8
y7
y6
y5 y3 b4
b3 Inte
nsity,
cp
s
0.5 1.0 1.5 0
1.8e7
0.99
Inte
nsity,
cp
s
200 400 600 800
Mass/Charge, Da
0
2.0e7
514.8
730.4 126.1 188.2 617.4
299.1 412.3 858.4 In
ten
sity,
cp
s
y8
y7 y6
546.4 y5 b2
b3
Click
for
more
info
© 2017 AB Sciex
• After MRM development and retention time
determination, next step is to tune the compensation
voltages for each peptide
• 3 step on column tuning strategy for large number of
peptides simultaneously
Three Step On-Column Tuning using Skyline with QTRAP® Systems
Streamlined, Iterative Tuning of Peptide CoV
5
10
15
20
25
30
Rough Tune
16.6 17.0
Time, min
0.0e0
1.0e4
2.0e4
3.0e4
4.0e4 16.8
Inte
nsity,
cp
s
19.4
19.6
19.8
20.0
20.2
20.4
20.6
Fine Tune
16.6 17.0
Time, min
0e0
1e4
2e4
3e4
4e4
16.8
Inte
nsity,
cp
s
17.0
18.0
19.0
20.0
21.0
22.0
23.0
Medium Tune
16.6 17.0
Time, min
0e0
2e4
4e4
16.8
Inte
nsity,
cp
s
Click
for
more
info
Thank You
© 2017 AB Sciex
For Research Use Only. Not for use in diagnostic procedures.
AB Sciex is doing business as SCIEX.
© 2017 AB Sciex. The trademarks mentioned herein are
the property of AB Sciex Pte. Ltd. or their respective owners.
AB SCIEX™ is being used under license.
Trademarks / Licensing