Native Protein Characterization Using the Agilent 6560 Ion Mobility Q-TOF

David Wong

Sr. Applications Scientist

June 2015

Confidentiality Label

1

Overview

• 6560 IM Q-TOF System Overview

• Benefits of Adding Ion Mobility to LC/Q-TOF/MS

• Carbohydrates and Protein Disulfide Bonds

• Protein Native vs. Denatured Conformation

• Antibodies (IgG, Herceptin and Biosimilar) Conformation

• Protein Complex

• Various IM Applications

• Recent Software (IM Browser) Development

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IM-QTOF Instrument Overview System sensitivity optimized using electrodynamic ion funnels to focus

and transmit ions

Ion Mobility resolution optimized while maintaining QTOF performance

(mass resolution and accuracy)

Ion Fragmentation can be selected using standard QTOF collision cell

(CID)

Bandwidth of QTOF data acquisition and processing channel was

increased by 10 fold to match the ion mobility data rates

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6560 Ion Mobility Q-TOF system design

Ionization source: Ion generation (ESI, AJS, Nano ESI, ChipCube, APCI etc.)

Front ion funnel: Efficient ion collection, desolvation and excess gas removal

Trap funnel: Ion accumulation and introducing ion packets into drift cell

Drift cell: Uniform low field ion mobility allows direct determination of accurate CCS (Ω)

Rear funnel: Efficient ion refocusing and introduction into mass analyzer

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IM Q-TOF/MS operational modes

• Mobility Separated Precursor Ion Mode

• Mobility Separated All Ions Fragmentation Mode

• Mobility Separated Targeted Precursor Ion Mode

• Mobility Separated Targeted MS/MS Mode

m/z selection

ON/OFF

Fragmentation

ON/OFF Mobility

separation

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Detector Analyte

Ions

Gating

Optics

Ion Mobility Cell VH VL

Electric Field

Stacked ring ion guide gives linear field

Basic operational principle of Ion Mobility for conventional

DC uniform field IMS

𝑣 = 𝐾 𝐸 ∝𝑒 𝐸

𝑃 𝑇 Ω

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Benefits of adding ion mobility to LC/Q-TOF/MS

Adds Additional Separation Power

• A new dimension of separation for increased mass spectral purity especially for complex mixture analysis

Improves Detection Limits

• Helps to eliminate interference from other analytes and background in the sample mixture

• Efficient ion focusing and transfer through the ion optics maximizes sensitivity for the overall system

Enhances Compound Identification

• Improves confidence in compound identification and ion structure

correlation through accurate collision cross section measurements

Provides Native Molecule Structural Information

• Differentiates various protein conformers (native vs. S-S mis-

matched, protein-protein interaction)

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Excellent in resolution and separation power

Chromatography Ion Mobility Mass

~ minutes ~ 60 milli-seconds ~ 100 m seconds

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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

Integrated Mass Spectrum:

Crude bacterial extract

(Prof. John McLean, Vanderbilt Univ.)

Ion mobility provides greater specificity

Mass (Da)

1444 1446 1450 1452 1454 1448

Mobility-Filtered Mass Spectrum:

S/N increased significantly!

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Resolving structural sugar isomers C18H32O16

Melezitose (DT: 25.76 ms)

Raffinose (DT: 26.68 ms)

Resolving two isomeric tri-saccharides

[M+Na]+

Carbohydrates analysis by IM-MS

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Ion

Mo

bilit

y D

rift

Tim

e (

ms)

Mass (Da) 0

0

20

40

50

500 1000 1500 2000

10

30

60 Mixture of Lacto-N-difucohexaose I & II

Mass (Da)

1018 1022 1024 1026 1028 1020

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

Oligosaccharide mixture

(Profs. John McLean and Jody May, Vanderbilt Univ.)

Carbohydrates -- Great complexity by linkage

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Source: Blixt et al., PNAS, 2004

4D (MS, DT, RT & TIC) Feature Finding or Library searches

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)

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

Siamycin II

Detecting miss-formed disulfide bonds: Siamycin II

(Profs. John McLean and Jody May, Vanderbilt Univ.)

Mass (Da)

1085 1086 1088 1089 1090 1087

[M+2H]2+

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Hemoglobin Conformation Analysis (at pH 2, pH 3.8 and pH 6.8)

September 3, 2015

Confidentiality Label

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pH 2

pH 3.8

pH 6.8

Heme-free α and Heme-free β

Heme-free α and Heme-bound α

Heme-bound α and Tetramer

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September 3, 2015

Confidentiality Label

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Deconveluted Results of Hemoglobin

Heme-free α

Heme-free β

Heme Heme-free α

Heme-bound α Heme-free β

Heme-bound α

Heme-free α ×2

Heme-free β ×2

Heme-free α ×2

Heme-free β ×2 Heme-bound α ×2

pH 2

pH3.8

pH6.8

pH 2

pH3.8

pH6.8

Change of Heme group

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IM Spectrum of Hemoglobin at pH 2 and pH 3.8

pH 2 Heme

pH 3.8

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y = 129.57x + 1774.9 R² = 0.9994

3200

3400

3600

3800

4000

4200

4400

4600

4800

5000

12 14 16 18 20 22 24

CCS values vs. Charge State

Series1 Linear (Series1)

pH 3.8

Collision Cross Section of Heme-free α Subunit

Charge

State m/z CCS (Å2)

13 1164.4 3452

14 1081.3 3569

15 1009.3 3730

16 946.3 3850

17 890.7 3988

18 841.2 4122

19 797 4249

20 757.2 4369

21 721.2 4490

22 688.6 4617

23 658.6 4748

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IM Spectrum of Hemoglobin at pH 7

Tetramer

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Drift Spectrum and CCS Values

Q15+

Q16+

Q17+ Q18+

Q19+

D10+/Q20+

4253 Å2

4421 Å2

4464 Å2 4512 Å2

Q15+

Q16+

Q17+

Q18+

Q19+

Q20+

Confirmation changing at different

charge state

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RF: 90 V

RF: 150 V

S1

S1 S2

S3

S4 S5

S1: Native

S2-S5: Denatured

• RF 90V

• RF 150V

S1 S2

S3

S4 S5

RF 90V RF 150V

IM analysis of cytochrom C (+8) (Uniform Drift Tube)

Preserve protein native structures!

– Due to the much lower ions

heating effects.

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All charge ions of IgG-2 under denatured condition (+45 to +70) posed the much smaller

drift times than the charge ions (+20 to +35) of native IgG-2.

IM Q-TOF/MS analysis of IgG-2 under the denatured and native conditions

IgG-2 (Denatured)

+60 +58

+56 +62

+64

IgG-2 (Native)

+26

+28

+24

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IM Q-TOF Comparison of IgG-1 and IgG-2 under native conditions

Native IgG-1

Native IgG-2 22+

22+

A

A

B

B

IgG-1 (22+)

IgG-2 (22+)

IgG-2 (22+ charge state) has more B isoform

mAb Structure: (Paul Schnier, Anal. Chem. 2010)

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IgG-1 Herceptin

IM Q-TOF/MS analysis of IgG-1 and Herceptin under the native condition

+22 +22

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IgG-1 posted slightly lower % of isoform B at its 22+ charge state. Overall, Herceptin has slightly larger

CCS values than IgG-1 with the same charge states.

Collision cross section (CCS) comparison of IgG-1 and Herceptin

IgG-1

Herceptin

+22

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IM Q-TOF comparison of Rituximab-1 (Innovator) and Rituximab-2 (biosimilar)

Rituximab-1

Rituximab-2

27+

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The average size of glycans on the Rituximab-1 were slightly smaller than those on the Rituximab-2. The

CCS of the 27+ molecule was larger for the Rituximab-2. Ion mobility can provide not only the size but also

the molecule structural information in the biosimilar study.

Collision cross section (CCS) comparison of Rituximab-1 (innovator) and Rituximab-2 (biosimilar)

Intact

Rituximab-1

Intact

Rituximab-2

27+

charge state

Rituximab-1

Rituximab-2

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Mass Spectrometric Analysis of Bovine Glutamate Dehydrogenase (GDH) Complex (Hexamer)

GDH is a hexamer of 500 residues with a molecular weight of ~56 kDa/each

337.65 kDa

Native condition

39+

40+

38+

41+

37+

27

40+

CCS: 11869 Å2

IM Q-TOF/MS analysis of bovine glutamate dehydrogenase (GDH) complex

(Hexamer)

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IM Q-TOF compendium of applications for 6560 IM Q-TOF

(5991-5723EN)

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Agilent’s high resolution discovery workflows Building blocks to drive analytical insight – B.07 release

4D Feature Finding Mass Profiler Agilent Ion Mobility

Q-TOF

Find Compare Acquire

• Two sample/group

compare of 4D Features

• Selected Features

displayed via Link to IM-

MS Browser*

• Feature ID via Link to ID

Browser

• Simple statistical

analysis (Student T,

PCA)

• CEF (w/o CCS) export

to MPP*

Identify

ID Browser &

PCDLs • View 4D Raw Data and

Detected Features

• Configurable

Abundance Maps

• Feature Lists linked to

selected IM frame and

zoomed region*

• Stepped Field CCS

Calculator and Easy

Single Field Calibration*

• Display and link high

and low collision energy

spectra

• Export pkl, mgf to

Spectrum Mill

• Stepped Field Method

• RT & m/z programmed

Quad Isolation

• Settable RF Trap

Parameters

• Alt. Gas Kit Support

• IM Multiplexing

• Alternating High/Low

Collision Energy

• MS1 (Quad) Isolation

• IM and QTOF SWARM

Autotune & IM Scope

• Molecular Formula

Generator

• Database Searching

• MS/MS Lib Search

(Qual DA via manual

export)

Characterize

IM-MS Browser

• Detects Ion Features

• Integrates total peak

Volume across RT, Drift

and Mass Dimensions

• Groups Features based

on isotopic pattern or

optionally leave

unassociated*

• Identifies charges state

and calculates and

annotates Features

with Collision Cross

Section (CCS)

Green in initial B.06 release

Plus Skyline….

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Summary

• Next generation of IM Q-TOF 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

The ‘Coolest’ Commercial Ion

Mobility System Available!

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Acknowledgements

Agilent Technologies:

Huy Bui, PhD.

Crystal Cody, Ph.D.

Ed Darland, Ph.D,

John Fjeldsted, Ph.D.

Chris Klein, Ph.D.

Ruwan Kurulugama, Ph.D.

Friedrich Mandel, Ph.D.

Sheher Mohsin, Ph.D.

Alex Mordehai, Ph.D.

Gregor Overney, PhD.

George Stafford, Ph.D.

Joachim Thiemann, Ph.D.

Bruce Wang

Agilent Collaborators:

John McLean, Ph.D., Vanderbilt University

Jody May, Ph.D., Vanderbilt University

Cody Goodwin, Ph.D., Vanderbilt University

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Ion mobility Q-TOF comparison

LC Drift IMS MS and MS/MS

High Resolution

Accurate Mass

Feature Uniform Ion Mobility (Agilent) Travelling Wave Ion Mobility Drift Mobility

advantage

Mobility

Resolution

Highest (can be > 80)

80cm drift tube (L)

Higher voltage (E)

No RF fields, Uniform low DC field

Generally around 30

10cm drift in TriWave,

Multi-section device

RF fields

Over 2X the IM

resolution of T-wave

Sensitivity High efficiency ion funnels - trapping

and rear

Step wave lens

Pressure barrier between Q and

TriWave

10X to 50X better than

T-wave

Collision Cross

Section (CCS)

measurement (Ω)

Direct determination of Ω

Low electric field and constant drift

tube pressure

Ω cannot be directly determined from

drift time.

Need calibration tables.

1-2% precision

Much better than

Synapt (>5%)

Molecular

structures

Lower RF fields, less ion heating. Higher RF fields, tendency for higher

fragmentation and ion heating

Lower RF allows

preservation of

molecular structures

Uniform

Ion Mobility

LC Q IMS MS

High Resolution Accurate Mass

Travelling Wave

Ion Mobility

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