advantages of ion mobility q-tof for characterization of ... · 13 carbohydrates analysis by im-ms...
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Add a New Dimension to your
Research Capability with Agilent’s
New Drift Ion Mobility Q-TOF System
Advantages of Ion Mobility Q-TOF for Characterization of Diverse Biological Molecules
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• 6560 IM Q-TOF/MS System Design
• System Unmatched Resolution
• Addition Separation Power
• Enhanced Selectivity for Greater Sensitivity
• Various IM Q-TOF Applications
• Direct Collision Cross Section (CCS) Determination
• Summary
Overview:
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What Does Ion Mobility Bring to Mass Spectrometry?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
Preserves Native Molecular Structure
• Effectively minimize ion heating effects to maintain native molecular conformations.
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IM-QTOF Instrument Overview• 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
Next generation Ion Mobility QTOF
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DetectorAnalyte
Ions
GatingOptics
Ion Mobility CellVH VL
Electric Field
Stacked ring ion guide gives linear field
Basic Operational Principle of Ion Mobility:- For Conventional DC Uniform Field IMS
∝
Ω
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Excellent in Resolution and Separation Power
Chromatography Ion Mobility Mass
~ 100 seconds~seconds ~60 milli-seconds
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530.7876
531.2889
531.7900
Ion Mobility Resolution:
IM Resolution
∆
IM Resolution: 63.30
MS Accuracy: < 1 ppm
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Ion Mobility Resolution - Continued
Resolution = 84!
Zipper Peptide:
(Prof. David Russell, Texas A&M)
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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
4x10
00.51
1.52
2.53
3.54
4.519.441
17 17.5 18 18.5 19 19.5 20 20.5 21 21.5
6x10
00.10.20.30.40.50.60.70.80.91
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
Theoretical Plot
IMS Drift Separation
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Separation of Isomers: Citrate and IsocitrateIsocitrate (C6H8O7)
Citrate (C6H8O7)
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MMA & SA can be separated nicely by IMS.
Separation of Methylmalonic Acid (MMA) and Succinic Acid (SA):
SA (C4H6O4)
MMA(C4H6O4)
SA(Dimer)
MMA(Dimer)
MMA and SA are isomers which cannot be separated by traditional MS.
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Resolving Structural Sugar Isomers C18H32O16
Melezitose
Raffinose
Resolving two isobaric tri-saccharides
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Carbohydrates Analysis by IM-MSIo
n M
obili
ty D
rift T
ime
(ms)
Mass (Da)0
0
20
40
50
500 1000 1500 2000
10
30
60
Mixture of Lacto-N-di-fructose hexose I & II
Mass (Da)1018 1022 1024 1026 10281020
Drift Time (ms)37 39 40 41 42383635
Lacto-N-di-fructose hexose II
Drift Time (ms)37 39 40 41 42383635
Lacto-N-di-fructose hexose I
Lacto-N-di-fructose hexose I
Lacto-N-di-fructose hexose II
Gal GlcGal GlcNAc
FucFuc
Gal GlcGal GlcNAc
Fuc Fuc
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0
20
40
50
500 1000 1500 2000
10
30
Mob
ility
Drif
t Tim
e (m
s)
Mass (Da)
Mass (Da)
1085 1086 1088 1089 10901087
Ion Mobility Drift Time (ms)
30 31 39 40 413835 36 37343332
Siamycin II
Detecting Miss-formed Disulfide Bonds: Siamycin II
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Protein Digest, Dr. Erin Baker, PNNL
Ion Mobility Provides Greater Separation
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Lipid Analysis: Mixture of L-α-phosphotidylethanolamine (PE) Lipids
+2 lipids
+3 lipids
+4 lipids
+1 lipids
PE 19:N
PE 29:NPE 33:N
PE 34:NPE 36:N
PE 38:N
PE 46:N
PE 37:N
PE 40:NPE 44:N
PE 48:N
PE 59:NPE 61:N
PE 63:N
PE 90:NPE 92:N
PE 38:4PE 38:6PE 38:5 PE 38:3
PE 38:2PE 38:1
PE 38:8PE 38:7
PE 38:0
PE 37:1
Ion
Mob
ility
Drif
t Tim
e (m
s)
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0
20
40
50
500 1000 1500 2000
10
30
Mob
ility
Drif
t Tim
e (m
s)
Mass (Da)
Mass (Da)1444 1446 1450 1452 14541448
Integrated Mass Spectrum:
Crude bacterial extract (Prof. John McLean, Vanderbilt Univ.)
Ion Mobility Provides Greater Specificity
Mass (Da)1444 1446 1450 1452 14541448
Mobility-Filtered Mass Spectrum:
S/N increased significantly!
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Enable the extraction of ion series of interest - ‘a group of glycans’ from matrix for further study. (Prof. Cathy Costello, Boston University)
• RNAseB Native Glycans Anaysis:
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Ion Mobility Provides Greater Specificity
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RNaseB Native Glycans(MS data before ion extraction)
Background noise greatly reduced!
Ion Mobility Simplifies Complex Spectra
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Glycan obscured by matrix can be identified
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Ion Mobility of Polymeric Ink Dispersants
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All Overlaid Extracted MS Spectra & Ion Mobility Heat Map
Ion mobility can resolve overlappingcharge-state isotopes unresolved bymass resolution
Ion Mobility of Polymeric Ink Dispersants
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Ion Mobility of Diesel (Hydrocarbons) Sample
Enable the extraction of ion series of interest for further study
Background noise reduced significantly!
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GC-APCI/IMS-QTOF analysis on ASTM compound mixture:
Some Possible Isomer Structures:
RT: 4.3 min
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GC-APCI/IMS-QTOF analysis on ASTM compound mixture:
Ethylbenzene1,4-Dimethylbenzene - Para-Xylene
1,3-Dimethylbenzene - Meta-Xylene
1,2-Dimethylbenzene - Ortho-Xylene
RT: 3.4 min
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200 400 600 800 1000 1200 1400 1600Drift Time (ms) vs. m/z
15
20
25
30
IMS-MS for Proteomics: Transmembrane Spanning Peptides of HeLa digest
Low dtHigh dt
770 771 772 773 774Drift Time (ms) vs. m/z
22
23
24
25
26
27
28
29
High dt
Low dt
All IonsFragmentation
Low dt
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Sensitivity - Detection limit of spiked compound in UrineQ-TOF mode
1 nM
10 nM
50 nM
100 nM
1000 nM
IM mode
10000 nM
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Ion Mobility Provides Greater Sensitivity!
y = 59060x - 350.63R² = 0.9993
0
200000
400000
600000
0 2 4 6 8 10
Are
a R
espo
nse
Sample Amount (pg)
Linear Dynamic Range
Sensitivity: ~ 50 fg of Reserpine
Dynamic range: ~ 3 - 4 orders
0E+00
1E+06
2E+06
3E+06
4E+06
5E+06
6E+06
single
pul
se1
mst
rapp
ing
single
pul
se4
mst
rapp
ing
single
pul
se8
mst
rap
time
mul
ti-pu
lse4x
1 m
stra
ppin
g
mul
ti-pu
lse8x
1 m
stra
ppin
g
Sign
al in
tens
ity (A
.U.)
• Integrated signal intensity for tetrakis decyl
ammonium bromide ion vs. ion trapping time for
single pulse and multi-pulse experiments.
• Multiplexing experiments result in at least 10X higher
signal intensity possibly due to less space charge
effects and detector saturation issues.
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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
S1S2
S3
S4 S5
RF 90V RF 150V
IM Comparison on Cytochrom C (+8):(Uniform Drift Tube vs. Travelling Wave)
Preserve protein native structures better! – Due to the much lower ions heating.
Travelling WaveIM data
?
Neutral Condition
Denatured Condition
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Ion Mobility Q-TOF on Protein Isoform Analysis
1346 1347 1348 1349 1350Drift Time (ms) vs. m/z
28
30
32
34
36
38
Cry34AB
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Denaturated Condition:
Resolving Isoforms of IgG-2
mAb Structure: (Publication Paul Schnier)
Only 1 conformation detected
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IM Q-TOF Analysis of Native IgG-2: 26+
25+
24+
23+
27+
28+
22+29+21+
26+25+
24+
23+
27+
28+
29+30+ 22+ 21+
Two possible conformations
detected
B
A
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IM Q-TOF Analysis of Native IgG-1: 26+
25+
24+
23+
27+
28+
29+30+
32
B
A
Two possible conformations also detected
22+21+
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IgG-2 (22+ charge state) has more B form .
IgG-1
IgG-2
IM Q-TOF Comparison of IgG-1 and IgG-2:
22+
22+
A
A
B
B
IgG-1(22+)
IgG-2(22+)
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0273
760Torr
2
K0 = Reduced Ion MobilityT = TemperatureP = PressureL = Drift lengthV = Voltage Drop across drift regiontd = Drift time
Determining Cross Sectional Areas
Ω /
/
.
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
Molecular size
X-ray crystallography
Ion mobility (using Helium)
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AnalyteMeasured
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%
LiteratureCross-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
[%]
0.560.280.410.010.24
• High experimental precision(< 0.5% relative deviation)
• Agreement with literature(most < 0.5% deviation)
Collision Cross Section Benchmark - Tetraalkylammonium Salts--- Vanderbilt University
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Cross Section Calculation of Ubiquitin Charge States
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 forcompactstructures approximate
region forelongatedstructures
Reference: Koeniger and Clemmer J Am Soc Mass Spectrom 2007, 18, 322-331
(Å)
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Cross Section Calculation of Ubiquitin Charge StatesIon 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
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Carbohydrates -- Great complexity by linkage
Source: Blixt et al., PNAS, 2004
Current dominant strategies: MSn or Library searches
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Conformational Space Occupancy of BiomoleculesC
ollis
ion
Cro
ss S
ectio
n(Å
2 )
Mass (Da)
Hypothetical Ordering ofBiomolecular Classes
lipids
carbohydrates
peptides
oligonucleotides
(Prof. John McLean, Vanderbilt U.)
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A Word About Instrument Design
LC Drift IMSMS and MS/MSHigh Resolution Accurate Mass
LC Q IMSMS
High Resolution Accurate Mass
Feature Drift Mobility TriWave Drift MobilityAdvantage
Mobility Resolution
Highest (can be > 80)80cm drift tube (L)higher voltage (E)No RF fields, Uniform low DC field
Generally around 3010cm drift TriWave, Multi-section deviceRF fields
Over 2X the IM resolution of T-wave
Sensitivity High efficiency ion funnels -trapping and rear
Step wave lensPressure barrier between Q and TriWave
10X to 50X better than T-wave
Collision Cross Section (CCS) measurement (Ω)
Direct determination of ΩLow electric field and constant drift tube pressure
Ω cannot be directly determined from drift time.Need calibration tables.
1-2% precision
Much better than T-wave (5-10%)
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
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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
• Preservation of molecular structures
• Direct determination collision cross sections