Development of a New Uniform-Field Ion

Mobility Quadrupole Time-of-Flight Mass

Spectrometer

Ruwan Kurulugama, Alexander Mordehai, Nathan Sanders, Ed Darland, Gregor Overney, Christian Klein, Crystal Cody, Bill Barry, George Stafford, John Fjeldsted Life Science Group Agilent Technologies, Santa Clara, CA

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Acknowledgments

SCIENTISTS

Ruwan Kurulugama

Alex Mordehai

Chris Klein

George Stafford

Nathan Sanders

Crystal Cody

Mikhail Ugarov

Ken Imatani

Shane Tichy

Lester Taylor

John Fjeldsted

COLLABORATORS

PNNL

NIH

Texas A&M

Vanderbilt University

Boston University School of Medicine

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DESIGN ENGINEERS

Mark Werlich

Tom Knotts

Lane Howard

Bill Frazer

Gene Wong

Yevgeny Kaplun

Bill Barry

SOFTWARE ENGINEERS Ed Darland Gregor Overney Bruce Wang Huy Bui Robert Kincaid Robin Scheiderer

Santa Clara IM QTOF Team

3 ASMS 2013

What Does Ion Mobility Bring to Mass Spectrometry?

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Separation •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

Confirmation •Improves confidence in compound identification and ion structure correlation through accurate collision cross section measurements

IMS Q-TOF Instrument Overview

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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

Ion Mobility System Design

Ion source maintained at ground potential (no voltage offset) Uniform low static electric field drift ion mobility

• 80 cm long, approximately 20 V/cm with 4 Torr Nitrogen • Mobility resolution approaches theoretical limit • Minimizes ion excitation or heating (helps to maintain ion structures and

conformations) Uniform low field ion mobility allows direct determination of accurate CCS (Ω) Ion fragmentation using CID enables an all ion experiment with the precursor

ions separated by drift times and product ions analyzed by high resolution QTOF

Ion Mobility Hardware View

Ion source front funnel

Ion trapping funnel

Ion collection rear funnel

Ion funnel technology brings sensitivity

Uniform field drift cell 80 cm

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It’s All About Separation

Chromatography Ion Mobility Mass

minutes 60 milli-seconds 160 µ-seconds

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Resolving Structural Sugar Isomers C18H32O16

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melezitose

Resolving two isobaric trisaccharides

raffinose

m/z

drift

tim

e (m

s)

527

528 529

M+Na +

All Ion MS using 20 Volt Fragmentation Energy

Collective drift spectrum includes all ions generated from 3 compounds

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Reserpine

Colchicine Ondansetron

100 m/z 600

12

drif

t tim

e (m

s)

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Simultaneous separation and fragmentation for colchicine

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Drift Time Separated Fragmentation

Colchicine [M+H]+

100 m/z 600

12

drif

t tim

e (m

s)

40

Collision Cross Section Correlates with Structure

Drift tube IM provides a direct accurate method to calculate collision cross sections Ω

Variables: Temperature Pressure Drift voltage Drift tube length

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Compound Drift time (ms)

Ω (lit. Å2)

Ω (obs. Å2)

Ondansetron 20.09 172.7 171.0

Colchicine 23.03 196.2 196.2

Reserpine 29.89 254.3 249.1

Mason-Schamp equation

SA

MMA

Ion mobility separation of MMA and SA

CCS Value:MMA 152.3 Å

SA 167.8 Å

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m/z

10 5

Counts

Drif

t tim

e in

ms

Cou

nts

14

BSA Digest with LC Separation

tD = 20.26 ms m/z = 435.9099

Precursor ion spectrum tR = 14.36 min

m/z

drift

tim

e (m

s)

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BSA Digest All Ion Fragmentation with LC Separation

Total fragment ion spectrum 30 V

Drift separated fragment ion spectrum tD = 20.26 ms

m/z 10X y1

b2

y1

b4

y4

b5 y6

m/z = 435.9099 [HLVDEPQNLIK + 3H]3+

drift

tim

e (m

s)

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Conformers tD = 21.14 ms 23.08 ms

HSA Digest Targeted MS/MS with LC Separation

m/z = 547.3174 (HSA) [KVPQVSTPTLVEVSR + 3H]3+

y8 b10

b9 y7

y6

y5 y4

Y8++ y3 b3 y2

b2

21.1

4 23.0

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Total fragment ion spectrum 15 V

drift

tim

e (m

s)

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Structural studies of proteins possible with CCS calculation

[M+5H]5+ [M+6H]6+

[M+7H]7+

[M+8H]8+

[M+9H]9+

[M+10H]10+

[M+11H]11+

[M+12H]12+

[M+13H]13+

[M+14H]14+

5+ 6+

7+ 8+

9+

13+

14+

10+ 11+ 12+

m/z

drift

tim

e (m

s)

IM Ubiquitin Charge States

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IM Ubiquitin Charge States

drift

tim

e (m

s)

m/z

Cross Section Calculation of Ubiquitin Charge States

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1000 1500 2000 2500 3000

m/z 1713.9, [M+5H]5+

m/z 1428.4, [M+6H]6+

m/z 1224.4, [M+7H]7+

m/z 1071.6, [M+8H]8+

m/z 952.6, [M+9H]9+

m/z 857.5, [M+10H]10+

m/z 779.6, [M+11H]11+

m/z 714.7, [M+12H]12+

m/z 659.8, [M+13H]13+

m/z 612.8, [M+14H]14+

Automated collision cross section calculation without the use of calibration curves

approximate region for compact structures approximate

region for elongated structures

Reference: Koeniger et al. J Am Soc Mass Spectrom 2007, 18, 322-331

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Ion Charge State CCS experimental (Å2)

CCS literature (Å2)

[M+5H]5+ 5 1196 [M+6H]6+ 6 1431, 1658 [M+7H]7+ 7 1755, 1886 1910 [M+8H]8+ 8 1966 1990 [M+9H]9+ 9 2008 2090

[M+10H]10+ 10 2114, 2197 2200 [M+11H]11+ 11 2239, 2348 2340 [M+12H]12+ 12 2412, 2511 2480 [M+13H]13+ 13 2556, 2620 2600 [M+14H]14+ 14 2680, 2726

Reference: Bush et al., Anal Chem. 2010, 82, 9557-9565.

Automated collision cross section calculation without the use of calibration curves

Cross Section Calculation of Ubiquitin Charge States

Added dimension of separation based on size, charge and ion structure

Resolve and characterize complex samples using LC/IM/MS analysis while maintaining high sensitivity

Direct method to calculate accurate collision cross sections for added analytical confirmation

Summary

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