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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
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IMS QTOF - Overview
• Background
• Ion Mobility Basics
• Instrument & Software Overview
• Applications
• Software tools
• Ω
• Lipids
• Carbohydrates
• Peptides
• Proteins
• Summary
9/24/2013 2
ASMS 2013 Ion Mobility
Abstracts
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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
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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
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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…
9/24/2013 5
1872 - 1946
Mass Spectrograph
Aston & Thomson
1919
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Resolution Is Important!
Chromatographic Ion Mobility Mass
9/24/2013 6
~seconds ~60 milli-seconds ~ 100 seconds
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It’s All About Separation
Chromatography Ion Mobility Mass
9/24/2013 7
~seconds ~60 milli-seconds ~100 seconds
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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
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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
9/24/2013 9
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
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IMS Q-TOF Instrument Overview
9/24/2013 10
• 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
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K = Ion Mobility
E = Electric Field
T = Temperature
P = Pressure e = charge on ion Ω = Collisional Cross Section
9/24/2013 11
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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
9/24/2013 12
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The Agilent Ion Mobility System
9/24/2013 13
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
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Front-end Instrumentation
9/24/2013 14
Ion funnel technology drives sensitivity gain
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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
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New Agilent MassHunter IMS Browser
Visualizing Ion Mobility LC/MS Data
9/24/2013 16
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
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New Agilent Masshunter IMS Browser
Visualizing Ion Mobility LC/MS Data
9/24/2013 17
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
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New Agilent Masshunter IMS Browser
Visualizing Ion Mobility LC/MS Data
9/24/2013 18
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
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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
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530.7876
531.2889
531.7900
Ion Mobility Resolution
9/24/2013 20
IMS
Resolution
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Ion Mobility Resolution
9/24/2013 21
536.5960 536.9300
537.2644
Resolution = 84!
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Saturday Night Plenary – David Russell
Time-dependent monomerization of Gramicidin A
9/24/2013 22
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
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Resolving Stereoisomers
9/24/2013
α-glucose β-glucose
Ion mobility enables separation of glucose stereoisomers
23
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Resolving Structural Sugar Isomers C18H32O16
24
Melezitose
Raffinose
Resolving two isobaric trisaccharides
9/24/2013
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Reveal Greater Detail
All Ions
9/24/2013 25
Reserpine
Colchicine
Ondansetron
609.2800
400.1749
294.1597
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Reveal Greater Detail
All Ions
9/24/2013 26
184.1117
170.0960
212.1064
294.1597
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Reveal Greater Detail
All Ions
9/24/2013 27
400.1749
358.1644
341.1383
326.1385 282.1244
382.1644
310.1192
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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 ????
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Step 1 - Determination of Cross Sections
9/24/2013 29
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Step 2 – Select Peak of Interest
9/24/2013 30
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Step 3 – Right Click Selected Peak
9/24/2013 31
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Step 4 – Table, Ω, Plot…Colchicine
Ω
9/24/2013 32
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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
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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
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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)
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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
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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
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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)
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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)
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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:
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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
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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
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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)
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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
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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!
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Herceptin - mAb
9/24/2013 46
+53 +51
+52
2851.3
607
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Herceptin IMS-MS
9/24/2013 47
Single conformer
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Herceptin Glyco-forms
9/24/2013 48
1
1
2
2
3
3
4
4
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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.
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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.
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• 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