Advantages of Ion Mobility Q-TOF for Characterization of ... · 13 Carbohydrates Analysis by IM-MS...

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New Drift Ion Mobility Q-TOF System

Advantages of Ion Mobility Q-TOF for Characterization of Diverse Biological Molecules

• 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:

2

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.

3

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

4

5

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

6

Excellent in Resolution and Separation Power

Chromatography Ion Mobility Mass

~ 100 seconds~seconds ~60 milli-seconds

7

530.7876

531.2889

531.7900

Ion Mobility Resolution:

IM Resolution

∆

IM Resolution: 63.30

MS Accuracy: < 1 ppm

8

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

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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12

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

14

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

16

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)

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:

18

Ion Mobility Provides Greater Specificity

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

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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22

Ion Mobility of Diesel (Hydrocarbons) Sample

Enable the extraction of ion series of interest for further study

Background noise reduced significantly!

23

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

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.

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

28

29

Ion Mobility Q-TOF on Protein Isoform Analysis

1346 1347 1348 1349 1350Drift Time (ms) vs. m/z

28

30

32

34

36

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Cry34AB

Denaturated Condition:

Resolving Isoforms of IgG-2

mAb Structure: (Publication Paul Schnier)

Only 1 conformation detected

30

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

31

IM Q-TOF Analysis of Native IgG-1: 26+

25+

24+

23+

27+

28+

29+30+

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B

A

Two possible conformations also detected

22+21+

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)

37

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

39

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