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Page 1: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

Omics Applications

SelexION® Differential Mobility Spectrometry Technology

RUO-MKT-11-6677-A

Page 2: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 3: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 4: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 5: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 2017 AB Sciex

Lipidomics Applications

Page 6: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 7: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 8: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 9: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 10: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 11: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 12: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 13: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 14: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 15: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 16: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 17: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 18: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 19: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 20: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 2017 AB Sciex

Metabolomics Applications

Page 21: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 22: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 23: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 24: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 2017 AB Sciex

• SelexION® technology enabled accurate quantitation with typical detection

levels of <2nmol on column.

Separation of TCA Cycle Metabolites

Metabolomics Applications

Page 25: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 2017 AB Sciex

Separation of TCA Cycle Metabolites

Metabolomics Applications

Maleic acid Fumaric acid

Maleic acid Fumaric acid

DMS On

DMS Off

Page 26: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 27: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 2017 AB Sciex

Proteomics Applications

Page 28: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 29: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 30: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 31: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 32: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 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

Page 33: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

Thank You

Page 34: Omics Applications - SCIEX · 2020-03-12 · glycolysis/gluconeogenesis pathways, cannot be distinguished based on their m/z. •However, at increased separation voltage (SV), each

© 2017 AB Sciex

For Research Use Only. Not for use in diagnostic procedures.

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the property of AB Sciex Pte. Ltd. or their respective owners.

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