Ion Mobility

Technology for

LC/MS

Reveal Greater Detail

Lester Taylor Ph.D.

Director LC/MS Product Marketing

Shane Tichy Ph.D

R&D Project Manager

September 24, 2013

9/24/2013 1

IMS QTOF - Overview

• Background

• Ion Mobility Basics

• Instrument & Software Overview

• Applications

• Software tools

• Ω

• Lipids

• Carbohydrates

• Peptides

• Proteins

• Summary

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ASMS 2013 Ion Mobility

Abstracts

Drift Ion Mobility for LC-MS

Cross sectional areas

Complex Samples

Shape and Charge

Conformers

Isomers

9/24/2013 3

Chromatography, Mass Resolution & now Ion Mobility

ASMS Scientific Presentations: – Disease research

– Proteomics, Metabolomics, Lipidomics

– Natural Products

– Fundamental studies

Ion Mobility

MS

Pacific Northwest

Labs

Texas A&M

Vanderbilt University

Boston University

NIH

IM resolution

Higher IM sensitivity

Resolve complex samples

Direct measurement of Ω

Preserve molecular structures

9/24/2013 4

Solving Analytical Problems

• Enhance throughput, improve sensitivity and quantitation

• For large scale -omics studies PNNL

• Improving glycan analysis

• Disease research - Entamoeba Boston University

• Ion mobility fundamentals

• Study of metallo-protein structures Texas A&M

• Collisional cross section data (Ω)

• Mapping specific chemical classes – natural products

Vanderbilt University

• Separation of androgenic steroids not amenable to LC & MS

• Ω used to identify isobaric steroids NIH

• Characterization of trans membrane domains.

• Preservation of fragile protein folding structures Agilent

1905

Ion mobility theory

Paul

Langevin

1969

Transport of Ions in Gases

McDaniel & Mason

1997

Applications to clusters & biomolecules

Clemmer & Jarrold

2006

Synapt Triwave

G2 in 2009

G2S in 2011

2013

Agilent IMS QTOF

Ion Mobility – A Brief History…

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1872 - 1946

Mass Spectrograph

Aston & Thomson

1919

Resolution Is Important!

Chromatographic Ion Mobility Mass

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~seconds ~60 milli-seconds ~ 100 seconds

It’s All About Separation

Chromatography Ion Mobility Mass

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~seconds ~60 milli-seconds ~100 seconds

Mass Accuracy Does Not Equal Compound

Identification: Seven Golden Rules - Oliver Fiehn

Empirical formula is not unique above

mass m/z 100 (searching PubChem)

Number of formula at ChemSpider mass

search at m/z 400.3787

• 1 ppm mass error → 1742 entries

• 0 ppm mass error → 340 entries

Need additional physical information to

identify

• MS/MS spectra

• Physical properties such as:

• Chromatographic retention time

• Ion mobility cross section

8

Number of Database Entries

(Assuming Zero Mass Error)

9/24/2013

Aldicarb-sulfone (C7H14N2O4S)

[M+Na]+ = 245.056649

Acetamiprid (C10H11ClN4)

[M+Na]+ = 245.056445

mass is 0.2 mDa requires ~2,000,000 resolution

Separation of Isobaric Pesticides

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4 x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

19.441

17 17.5 18 18.5 19 19.5 20 20.5 21 21.5

6 x10

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

18.297

Drift Time (ms) 17 17.5 18 18.5 19 19.5 20 20.5 21 21.5

Aldicarb-sulfone

Acetamiprid

Drift Time (ms)

17 17.5 18 18.5 19 19.5 20 20.5 21 21.5

4x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

+IMS DriftSpec (m/z: 245.013827-245.177238) (rt: 0.026-1.987 min) Aldicarbsulfone_A…

* 18.297

* 19.441

Counts vs. Acquisition Time (min)17 17.5 18 18.5 19 19.5 20 20.5 21 21.5

Theoretical Plot

IMS Drift

Separation

IMS Q-TOF Instrument Overview

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• Based on 6550 Q-TOF

– Maintains QTOF performance

– Inserted trapping funnel and drift tube

– Trapping funnel & gate

– Matches IMS duty cycle with Q-TOF analysis

• Precursor ions are separated by drift time and

• Product ions are analyzed by QTOF.

Next generation Ion Mobility QTOF

K = Ion Mobility

E = Electric Field

T = Temperature

P = Pressure e = charge on ion Ω = Collisional Cross Section

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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

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The Agilent Ion Mobility System

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Electric Field

• Nitrogen buffer gas

• Funnels drive sensitivity

• Uniform Field Drift Tube allows direct determination of Ω

• Resolution approaches theoretical limit

• Fragmentation after IMS means parents and fragments have common drift times

Mobility Resolution

Front-end Instrumentation

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Ion funnel technology drives sensitivity gain

K0 = Reduced Ion Mobility

T = Temperature

P = Pressure

L = Drift length

V = Voltage Drop across drift region

td = Drift time

Determining Cross Sectional Areas?

Boltzmann constant

Charge on

an electron

Charge state of the

analyte ion

Reduced mass of

the ion and neutral

Number density

of the drift gas

Electric field

15 9/24/2013

New Agilent MassHunter IMS Browser

Visualizing Ion Mobility LC/MS Data

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322.0481

622.0294

922.0098 1221.9906 1521.9711

1821.9521 2121.9332

2421.9138

2721.8941

Ion mobility Frame Viewer provides linked views for easier navigation

IMS/MS Frame

Selection

Chromatogram View

Software

Solutions for

Improving your

Productivity

New Agilent Masshunter IMS Browser

Visualizing Ion Mobility LC/MS Data

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Frame View

Ion mobility Frame Viewer provides linked views for easier navigation

322.0481

622.0294

922.0098 1221.9906 1521.9711

1821.9521 2121.9332

2421.9138

2721.8941

Easily

Customized

for your

Preference

New Agilent Masshunter IMS Browser

Visualizing Ion Mobility LC/MS Data

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Ion mobility Frame Viewer provides linked views for easier navigation

322.0481

622.0294

922.0098 1221.9906 1521.9711 1821.9521

2121.9332

2421.9138

2721.8941

New Agilent Masshunter IMS Browser

Visualizing Ion Mobility LC/MS Data

9/24/2013 19

Ion mobility Frame Viewer provides linked views for easier navigation

2721.8941

2722.8974

2723.9003

530.7876

531.2889

531.7900

Ion Mobility Resolution

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IMS

Resolution

Ion Mobility Resolution

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536.5960 536.9300

537.2644

Resolution = 84!

Saturday Night Plenary – David Russell

Time-dependent monomerization of Gramicidin A

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12 13 14 15 16 17 18 19 20 21 220.0

0.2

0.4

0.6

0.8

1.0

0 min

177 min

299 min

451 min

1707 min

Re

lati

ve

ab

un

da

nc

e

Drift time (ms)

D1

D2

D3

Dimer

[2GA + 2Na]2+

Monomer

[GA + Na]+

(B)

D2

D2’

Monomer

[GA + Na]+

D1

D3

Drift Time (ms)

Synapt Agilent IMS

Resolving Stereoisomers

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α-glucose β-glucose

Ion mobility enables separation of glucose stereoisomers

23

Resolving Structural Sugar Isomers C18H32O16

24

Melezitose

Raffinose

Resolving two isobaric trisaccharides

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Reveal Greater Detail

All Ions

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Reserpine

Colchicine

Ondansetron

609.2800

400.1749

294.1597

Reveal Greater Detail

All Ions

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184.1117

170.0960

212.1064

294.1597

Reveal Greater Detail

All Ions

9/24/2013 27

400.1749

358.1644

341.1383

326.1385 282.1244

382.1644

310.1192

Collision Cross Section Correlates with Structure

Drift tube IMS provides a direct method to calculate collision cross sections Ω:

Variables: Temperature

Pressure

Drift voltage

Drift tube length

9/24/2013 28

Compound Drift time

(ms) Ω

(lit. Å2)

Ω (obs. Å2)

Ondansetron 20.09 172.7 ????

Colchicine 23.03 196.2 ????

Reserpine 29.89 254.3 ????

Step 1 - Determination of Cross Sections

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Step 2 – Select Peak of Interest

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Step 3 – Right Click Selected Peak

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Step 4 – Table, Ω, Plot…Colchicine

Ω

9/24/2013 32

Published Collisional Cross Sections

Analyte Mass

[Da]

CCS

Literature

[Å2]

CCS

This Work

[Å2]

%

Deviation

from Lit.

Colchicine1 399.4 196.2 196.2 ± 0.54 Å2 0%

Odansetron2 293.4 172.7 173.8 ± 0.36 Å2 0.6%

Threonine 119.1 130.1 ±0.45 Å2 <2%

Phenylalanine 165.2 140.9 ±0.5 Å2 <2%

Tyrosine 181.2 148.4 ±0.6 Å2 <2%

Fructose 180.2 143.4 ±0.6 Å2 <2%

Sorbitol 182.2 142.7 ±0.5 Å2 <2%

1. Anal.Chem. 2012;84:1026.

2. Int. J. MS. 2010;298:78

3. JASMS.2007;18:1163

New Analyte Ion

CCS

IMS QTOF

[Å2]

5α-dihydrotestosterone (M+H)+ 181.6 ± 0.

5α-dihydrotestosterone (M+Na)+ 201.5±1.0

5β-dihydrotestosterone (M+H)+ 179.8±0.8

5β-dihydrotestosterone (M+Na)+ 199.5±0.8

androsterone (M+Na)+ 200.0±0.7

etiocholanolone (M+Na)+ 196.3±1.1

5-androstenediol (M+Na)+ 174.0±1.5

epiandrosterone (M+Na)+ 197.0±0.8

Excellent agreement between published

and measured cross sections

9/24/2013

Collaboration with NIH

33

Collision Cross Section Benchmark

Vanderbilt University

9/24/2013 34

• Tetraalkylammonium salts (TAA)

• Proposed as an “ideal” ion mobility standard

• Wide CCS range (TAA-4 to TAA-18; 100 to 400 Å2)

• TAA salts do not form clusters

• Literature CCS values exist N2 drift gas

+2 ions

+3 ions

+1 ions TAA-16

TAA-18

TAA-12

TAA-10

TAA-8 TAA-7

TAA-6 TAA-5

TAA-4

0

10

30

40

200 400 600 800

0

20

Mo

bilit

y D

rift

Tim

e (

ms)

Mass-to-Charge (m/z) 1000 1200

50

TAA-5 N-(CH2CH2CH2CH2CH3)4

Tetraalkylammonium Salts CCS Values Compared to Literature

9/24/2013 35

Analyte

Measured

Cross-Section

[Å2]

TAA-4 166.61 ± 0.5%

TAA-5 189.21 ± 0.6%

TAA-6 212.71 ± 0.3%

TAA-7 236.34 ± 0.2%

TAA-8 257.19 ± 0.1%

TAA-10 294.53 ± 0.1%

TAA-12 323.62 ± 0.2%

TAA-16 362.03 ± 0.2%

TAA-18 381.58 ± 0.3%

Literature

Cross-Section

[Å2]

166.00 ± 0.3%

190.10 ± 0.1%

214.00 ± 0.3%

236.80 ± 0.2%

258.30 ± 0.4%

Relative Standard

Deviation

[%]

0.56

0.28

0.41

0.01

0.24

• High experimental precision

(< 0.5% relative deviation)

• Agreement with literature

(most < 0.5% deviation)

Conformational Space Occupancy of Biomolecules

9/24/2013 36

Co

llis

ion

Cro

ss

Se

cti

on

(Å

2)

Mass (Da)

Hypothetical Ordering of

Biomolecular Classes

lipids

carbohydrates

peptides

oligonucleotides

Lipid Analysis

9/24/2013 37

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

9/24/2013 38

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

9/24/2013 39

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 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)

Crude Bacterial Extract

9/24/2013 40

0

20

40

50

500 1000 1500 2000

10

30

Mo

bilit

y D

rift

Tim

e (

ms)

Mass (Da)

Mass (Da)

1444 1446 1450 1452 1454 1448

Mass (Da)

1444 1446 1450 1452 1454 1448

Integrated Mass Spectrum:

Mobility-Filtered Mass Spectrum:

9/24/2013 41

0

20

40

50

500 1000 1500 2000

10

30

Mo

bilit

y D

rift

Tim

e (

ms)

Mass (Da)

Ion Mobility Drift Time (ms)

18 22 54 58 62 50 38 42 46 34 30 26

R = 72.1

Extracted Mobility Spectrum:

Crude Bacterial Extract

Crude Bacterial Extract

9/24/2013 42

0

20

40

50

500 1000 1500 2000

10

30

Mo

bilit

y D

rift

Tim

e (

ms)

Mass (Da)

Mass (Da)

1085 1086 1088 1089 1090 1087

Ion Mobility Drift Time (ms)

30 31 39 40 41 38 35 36 37 34 33 32

siamycin II

Carbohydrates IM-MS

9/24/2013 43

maltodextrins (1 to 8) cyclodextrins (α, β, γ)

human milk oligosaccharides (7)

TAA Salts

+2 ions

+3 ions

+1 ions

+4 ions

Ion

Mo

bil

ity D

rift

Tim

e (

ms)

0

0

20

40

50

500 1000 1500 2000

10

30

60

Mass-to-Charge (m/z)

Carbohydrates IM-MS

9/24/2013 44

Ion

Mo

bilit

y D

rift

Tim

e (

ms)

0

0

20

40

50

500 1000 1500 2000

10

30

60 Mixture of Lacto-N-difucohexaose I & II

1018 1022 1024 1026 1028 1020

Mass-to-Charge (m/z)

Mass-to-Charge (m/z)

Lacto-N-difucohexaose I

Lacto-N-difucohexaose II

Gal Glc Gal GlcNAc

Fuc Fuc

Gal Glc Gal GlcNAc

Fuc Fuc

Drift Time (ms)

37 39 40 41 42 38 36 35

Lacto-N-

difucohexaose II

Drift Time (ms)

37 39 40 41 42 38 36 35

Lacto-N-

difucohexaose I

IMS-MS for Proteomics: Transmembrane Spanning

Peptides

Characteristics:

• Large hydrophobic peptides

• X-ray: 7-helical domains

• Confirmation impacts function of membrane

• Membrane proteins are drug targets

Challenge:

• Detect conformational changes that may be related to function

• Isoforms not separated by LC/MS

September 24, 2013 45

Tuesday Poster TP-647

600 800 1000 1200 1400 1600 1800

Drift Time (ms) vs. m/z

20

25

30

35

40

45

50

1110.5 1111.0 1111.5 1112.0 1112.5

Drift Time (ms) vs. m/z

32

34

36

38

40

42

1502.5 1503.0 1503.5 1504.0 1504.5 1505.0

Drift Time (ms) vs. m/z

34

36

38

40

42

44

m/z Mass Seq. Loc. CCS [Å2] Std Dev

1211.172 4840.6568 A(83-129) 1166.9 3.46

494.3194 986.6248 A(217-225) 346.3 1.80

1110.607 3328.8003 A(1-30) 833.9 3.14

1104.587 4414.3174 A(1-41) 1017.6 2.91

959.9341 1917.8536 A(228-248) 465.8 3.69

726.8591 1451.702 A(160-172) 407.5 1.66

1081.498 2160.9822 A(226-248) 479.7 2.29

1429.742 4286.205 A(1-40) 966.2 4.34

1429.742 4286.205 A(1-40) 846.5 1.44

1502.943 9011.6045 A(1-82) 1723.2 6.76

1502.943 9011.6045 A(1-82) 1610.4 3.47

Isoforms peptides identified by drift separation!

Herceptin - mAb

9/24/2013 46

+53 +51

+52

2851.3

607

Herceptin IMS-MS

9/24/2013 47

Single conformer

Herceptin Glyco-forms

9/24/2013 48

1

1

2

2

3

3

4

4

Resolving Isoforms of IgG2

9/24/2013 49

D. Bagal, J. F. Valliere-Douglass, A. Balland, P. D. Schnier. “Resolving Disulfide Structural Isoforms of IgG2 Monoclonal Antibodies by Ion Mobility Mass

Spectrometry” Anal. Chem. 2010, 82, 6751-6755.

9/24/2013 50

Resolving Isoforms of IgG2

1000 2000 3000 4000 5000 6000

Drift Time (ms) vs. m/z

0

20

40

60

80

100

4 x10

0

0.5

1

1.5

2

2.5

3

3.5

4

+IMS Drift Spectrum (0.00-106.65 ms) …

Counts vs. Drift Time (ms)

10 20 30 40 50 60 70 80 90 100

1000 2000 3000 4000 5000 6000

Drift Time (ms) vs. m/z

0

20

40

60

80

100

New Agilent IMS

D. Bagal, J. F. Valliere-Douglass, A. Balland, P. D. Schnier. “Resolving Disulfide Structural Isoforms of IgG2 Monoclonal Antibodies by Ion Mobility Mass

Spectrometry” Anal. Chem. 2010, 82, 6751-6755.

• Next generation of IMS QTOF Technology

• Added dimension of separation based on size, charge and molecular

conformation

• Resolve and characterize the complex samples

– Increased peak capacity

• Direct determination collision cross sections

• Preservation of molecular structures

Summary

9/24/2013 51