Native Protein Characterization Using the Agilent 6560 Ion Mobility Q-TOF
David Wong
Sr. Applications Scientist
June 2015
Confidentiality Label
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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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