advantages of ion mobility qtof for characterization of large … · 2016-09-11 · 30 31 32 33 34...

Add a New Dimension to your

Research Capability with

Agilent’s New Drift Ion Mobility

QTOF System

Ning Tang, Ph.D. Application Scientist

Anne Blackwell, Ph.D. Application Scientist

Advantages of Ion Mobility QTOF for

Characterization of Large Molecules

2014 Biopharma Roadshow 1

IM-QTOF Instrument Overview

2

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

2014 Biopharma Roadshow

Ion Mobility System Design

3

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


4

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


5

Resolution Is Important!

Chromatographic Ion Mobility Mass

~seconds ~60 milli-seconds ~ 100 m seconds


6

530.7876

531.2889

531.7900

Ion Mobility Resolution

IMS

Resolution

𝑹 =𝒕𝒅

∆𝒕𝒅

=𝑳𝑬𝑸

𝟏𝟔𝒌𝑻𝒍𝒏𝟐


//upload.wikimedia.org/wikipedia/commons/5/51/Bradykinin_updated.png

536.5960 536.9300

537.2644

7

Ion Mobility Resolution - Continued

Resolution = 84!


8

It’s All About Separation

Chromatography Ion Mobility Mass


~seconds ~60 milli-seconds ~ 100 m seconds

9

Chromatography Ion Mobility Mass

Peak Capacity = IMResolution x MassResolution x Orthogonality

Peak Capacity = 60 x 40,000 x 14% = 336,600 ~ 8-fold increase

Dwivedi P, Schultz AJ, Hill HH,

Metabolic profiling of human blood by high-resolution ion mobility mass spectrometry (IM-MS).

Int J Mass Spectrom 298:78–90. 2010

It’s All About Separation


10

Aldicarb-sulfone (C7H14N2O4S)

[M+Na]+ = 245.056649

Acetamiprid (C10H11ClN4)

[M+Na]+ = 245.056445

D mass is 0.2 mDa requires ~ 2,000,000 resolution !

Separation of Isobaric Pesticides

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


//upload.wikimedia.org/wikipedia/commons/4/4a/Acetamiprid_Structural_Formulae_V.1.svg

//upload.wikimedia.org/wikipedia/commons/f/f5/Aldicarb-2D-skeletal.png

11

Resolving Structural Sugar Isomers C18H32O16

Melezitose

Raffinose

Resolving two isobaric tri-saccharides


12

Crude Bacterial Extract

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


Working Hypothesis:

α-helical membrane spanning domains stay intact, and differ in drift-time from denatured peptides

400 600 800 1000 1200 1400 1600Drift Time (ms) vs. m/z

20

25

30

35

40

45

50

55

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

1502.5 1503.0 1503.5 1504.0 1504.5 1505.0

Drift Time (ms) vs. m/z

34

36

38

40

42

44

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

30

35

40

45

50


13

IMS-MS for Proteomics: Transmembrane Spanning Peptides

1429.5 1430.0 1430.5 1431.0 1431.5 1432.0


34

36

38

40

42

44

1502.5 1503.0 1503.5 1504.0 1504.5 1505.0


34

36

38

40

42

44

1694.5 1695.0 1695.5 1696.0


44

45

46

47

48

49

50

51

52

53

1346.5 1347.0 1347.5 1348.0


34

36

38

40

42

44

46

A B

C D

Figure Seq. Loc. Chg. State Helix A 1-40 [M+3H]3+ A B 1-82 [M+6H]6+ A-B C 83-159 [M+5H]5+ C-D-E

D 176-225 [M+4H]4+ F-G


14

15

LC – Ion Mobility – QTOF Protein Isoform Analysis

1346 1347 1348 1349 1350


28

30

32

34

36

38


Cry34AB

Resolving Isoforms of IgG2

16


17

Resolving Isoforms of IgG2

Publication Paul Schnier, Synapt:

3uM direct infusion in 160 mM NH4Ac

1000 2000 3000 4000 5000 6000


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


0

20

40

60

80

100


18

Why Ion Mobility? Separation!

Protein Digest, Erin Baker, PNNL


19

Lipid Analysis: Mixture of L-α-phosphotidylethanolamine (PE) Lipids

+2 lipids

+3 lipids

+4 lipids

+1 lipids

PE 19:N

PE 29:N PE 33:N

PE 34:N PE 36:N

PE 38:N

PE 46:N

PE 37:N

PE 40:N PE 44:N

PE 48:N

PE 59:N PE 61:N

PE 63:N

PE 90:N PE 92:N

PE 38:4 PE 38:6 PE 38:5 PE 38:3

PE 38:2 PE 38:1

PE 38:8 PE 38:7

PE 38:0

PE 37:1

Ion

Mo

bilit

y D

rift

Tim

e (

ms

)


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:

Why Ion Mobility? Specificity!

Crude bacterial extract, John McLean, Vanderbilt


20

21


Why Ion Mobility? Sensitivity!

y = 59060x - 350.63 R² = 0.9993

0

200000

400000

600000

0 2 4 6 8 10

Are

a R

esp

on

se

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

puls

e

1 m

str

appin

g

single

puls

e

4m

str

appin

g

single

puls

e

8 m

str

ap t

ime

mult

i-puls

e

4x1

ms

trappin

g

mult

i-puls

e

8x1

ms

trappin

g

Sig

nal in

tensi

ty (

A.U

.)

• Integrated signal intensity for tetrakis decyl

ammonium bromide ion versus ion trapping time

for single pulse and multi-pulse experiments.

• These data indicate that for the same amount of

trapping time, multiplexing experiments result in at

least 10X higher signal intensity possibly due to

less space charge effects and detector saturation

issues.

Data browser can be used to “isolate” a group of glycans from matrix

Isolation of RNAseB native Glycans, Cathy Costello, Boston University

Why Ion Mobility? Selectivity!


22

Simplified view of RNaseB native

glycan

RNaseB native glycans

Glycan obscured by matrix is identified after cleaning up

background chemical noise using ion mobility

Why Ion Mobility? Selectivity!


23

Ion Mobility of Polymeric Ink Dispersants


24

𝐾0 =273 𝐾

𝑇

𝑃

760 Torr

𝐿2

𝑉𝑡𝑑

K0 = Reduced Ion Mobility

T = Temperature

P = Pressure

L = Drift length

V = Voltage Drop across drift region

td = Drift time

Determining Cross Sectional Areas

Ω = (18𝞹)1/2

16

𝑧𝑒

(𝑘𝑏𝑇)1/2 [1

𝑚𝑖

+1

𝑚𝐵

]1

2 𝑡𝑑

𝐸

𝐿

760

𝑃

𝑇

273.2

1

𝑁

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)


25

26

Standard Procedure for Calculating CCS (Auto Charting, Curve Fitting and Calculating the CCS)


Time segments with different drift fields

27

Collision Cross Section Benchmark --- Vanderbilt University

• 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


28

Tetraalkylammonium Salts

--- CCS Values Compared to Literature

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)


29

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 for

compact

structures approximate

region for

elongated

structures

Reference: Koeniger and Clemmer J Am Soc Mass Spectrom 2007, 18, 322-331

(Å)


30

Cross Section Calculation of Ubiquitin Charge States

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


31

Carbohydrates -- Great complexity by linkage

Source: Blixt et al., PNAS, 2004

Current dominant strategies: MSn or Library searches


32

Carbohydrates IM-MS Io

n M

ob

ilit

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

Lacto-N-difucohexaose I

Lacto-N-difucohexaose II

Gal Glc Gal GlcNAc

Fuc Fuc

Gal Glc Gal GlcNAc

Fuc Fuc


33

IM Drift Spectrum on Cytochrome C at various RFs:

S1: Native

S2-S5: Denatured

• RF 90V

• RF 110V

• RF 130V

• RF 150V

• RF 180V

Waters

Synapt G2

IM data

S1

S2

S3

S4 S5

?

Preserve protein native

structures better! – Due

to the much lower ions

heating.


34

200 400 600 800 1000 1200 1400 1600


15

20

25

30

IMS-MS for Proteomics: Transmembrane Spanning Peptides of HeLa digest

Low dt High dt


770 771 772 773 774


22

23

24

25

26

27

28

29

High dt

Low dt

All Ions

Fragmentation

Low dt

35

A Word About Instrument Design

LC Drift IMS MS and MS/MS

High Resolution Accurate Mass

LC Q IMS MS

High Resolution Accurate Mass

Feature Agilent Waters 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

25cm drift 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 (5-10%)

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


36

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


2014 Biopharma Roadshow 37

Acknowledgments

Ruwan Kurulugama

Alex Mordehai

Mark Werlich

Chris Klein

Tom Knotts

Ed Darland

Gregor Overney

Bill Barry

Nathan Sanders

Lane Howard

Mikhail Ugarov

Gene Wong

Yergeny Kaplun

George Stafford

Bill Frazier

Bruce Wang

Huy Bui

Crystal Cody

John Fjelsted

Ken Imatani

Robert Kincaid

Robin Scheiderer

Collaborators:

PNNL

NIH

Texas A&M

Vanderbilt University

Boston University

38


Standard Procedure for Calculating CCS (Drift Time versus P/V Curves)

Reserpine Colchicine

Ondansetron y = 6258x + 5.5225R² = 0.9999

y = 8,019x + 7.2356R² = 0.9996

y = 5,410x + 4.9269R² = 1.0000

0

10

20

30

40

50

60

0 0.001 0.002 0.003 0.004 0.005 0.006D

rift

tim

e (m

s)P/V (Torr/V)

Colchicine Reserpine Ondansetron

39

Compound Drift time

(ms) Slope

Intercept

t0(ms)

Ondansetron 20.09 5410 4.9269

Colchicine 23.03 6258 5.5225

Reserpine 29.89 8019 7.2356

tD = experimental drift time

t0 = time ions spend out side

the drift cell

td = corrected drift time


39

40

Standard Procedure for Calculating CCS

- Auto Stepping the Low Field Gradient

Time segments with different drift fields


41

Conformational Space Occupancy of Biomolecules

C

oll

isio

n C

ros

s S

ec

tio

n (

Å2)

Mass (Da)

Hypothetical Ordering of

Biomolecular Classes

lipids

carbohydrates

peptides

oligonucleotides


42

Ion source maintained at ground potential (no voltage offset)

Uniform low static electric field across drift cell

• 80 cm long, approximately 20 V/cm with 4 Torr Nitrogen buffer gas

• 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)

~1700 V on chamber

Desolvation

chamber at 0 V

Uniform DC electric field

(no RF voltage applied)

Ion Mobility System Design


43

Uniform field

drift cell 80 cm

43

Ion funnel technology improves sensitivity

Drift Cell

Front funnel Trapping funnel Drift Cell (80 cm) Rear funnel

Ion Mobility Hardware Design


advantages of ion mobility qtof for characterization of large … · 2016-09-11 · 30 31 32 33 34...

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