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The Measure of Confidence

ADD A NEW DIMENSION TO YOUR RESEARCH WITH THE AGILENT 6560 ION MOBILITY Q-TOF LC/MS

Applications Highlights

The Agilent 6560 Ion Mobility Q-TOF LC/MS system is the fi rst commercially

available uniform fi eld ion mobility system. When coupled with the Agilent Infi nity

II UHPLC system, it provides a new dimension of separation power combining

the selectivity of liquid chromatography, ion mobility, and mass spectrometry

techniques.

Laboratories can accelerate research and gain greater confi dence in compound

identifi cation with the additional dimension of mobility separation and collision

cross section, as well as the structural information provided by ion mobility

measurements. This instrument simultaneously provides high sensitivity

and accurate collision cross section measurements. This document provides

an overview of the technology and examples of real-world applications to

demonstrate its capabilities.

What is Ion Mobility?Principles of ion mobility separationIn a classical uniform fi eld drift tube, the electric fi eld within the drift cell moves

ions through the device while the drag force acts against the electrical force that

moves the ions, due to the collisions of these ions with the stationary buffer gas

molecules. The drag force experienced by the ions depends on their collision cross

sections (a function of size and shape), electrical charge, and mass. Multiple

charged ions move through the buffer gas more effectively than single charged

ions, because they experience a greater force due to the electric fi eld. Ions with

larger cross sections are slowed more easily by collisions with the buffer gas

in the drift tube. The drag force resulting from collisions of ions with buffer gas

molecules acts against their acceleration. Thus, an equilibrium state is quickly

reached, and the ions start moving with constant velocity (V ), proportional to the

applied electric fi eld (E). The proportionality constant (K) is the gas phase mobility

of an ion. This process can be expressed in the equation: V = KE.

Ken Imatani

Agilent Q-TOF LC/MS

Product Manager

David Wong, Ph.D.

Senior Application Scientist

2

Figure 1. Schematic of an Agilent uniform fi eld drift tube coupled to a quadrupole time-of-fl ight mass

spectrometer using a hexapole ion guide. The Q-TOF MS has a mass resolution of over 40,000, and fi ve orders of

magnitude dynamic range in Q-TOF mode of operation.

Front funnel

Trapping gate

Drift tube

Ionization source

Trapping funnel

Rear funnel

Quad mass filter

Collision cell

Ion pulser

IBC

Finding and confi rming minor components• Readily detect low femtogram

analytes in complex matrices using

electrodynamic funnel technology.

• Confi dently identify compounds

using All Ions MS/MS.

Preserving protein conformations • Easily study gas phase peptide and

protein structures.

• Effectively minimize ion heating

effects to maintain molecular

conformations.

The Agilent 6560 ion mobility system

can provide researchers with greater

analytical detail for the following

challenges:

Resolving structural isomers• Probe the molecular structure

and conformation of peptides and

proteins using high-resolution ion

mobility separation.

• Directly determine molecular size

(from collision cross sections)

without reference standards or

calibration tables.

Increasing peak capacity • Effectively resolve individual

components in complex mixtures

with the combined power of UHPLC,

ion mobility, and mass spectrometry.

• Obtain optimal ion mobility

separation with double-grid trapping

technology.

Mobility is a function of an ion’s

interaction with the buffer gas,

its mass, and its electrical charge.

Furthermore, mobility depends on the

gas temperature and the mass of the

buffer gas molecules.

• L is the length of the drift cell

• td is the corrected drift time

• E is the electric fi eld across the drift

cell

• P is the pressure of the drift cell

• T is the temperature of the buffer

gas

Why Ion Mobility? Achieve greater analytical detail for complex samplesThe 6560 system was developed with

the collaboration of scientists from

a number of academic institutions

and government laboratories. In

multiple studies, the instrument has

demonstrated the ability to reveal

signifi cantly greater analytical detail

for complex samples, compared to

high resolution mass spectrometry

technology alone.

Researchers have reported that while

high resolution mass spectrometry has

become the analytical cornerstone for

proteomics, metabolomics, and other

research applications requiring the

analysis of highly complex samples,

there has also been signifi cant interest

in the use of ultra-fast orthogonal

techniques to provide added

dimensions of separation.

K0 = L

tdEP

760

273.2

T

3

Applications in this document

demonstrate that the instrument

delivers:

• Greater separation of lipids and

glyco-peptides

• More accurate collision cross

section measurements, enabling

more confi dent characterization

of structural conformations and

isomeric compounds

• Greater numbers of trace level

peptides in complex matrices

• Preservation of structural fi delity of

metallo-proteins in solutions

To maximize the analytical utility

of this system, Agilent has also

developed software tools for the

visualization of ion mobility data.

Agilent MassHunter Software is

designed to allow researchers to

interrogate mobility/mass domain

data, and easily determine collisional

cross section values with high

precision and accuracy. Agilent also

provides advanced browsing capability

and feature fi nding tools to take

advantage of the mobility data.

Agilent Ion Mobility: higher quality MS/MS spectra at trace levelsThe Agilent 6560 Ion Mobility Q-TOF

LC/MS system enables direct

collision cross section (CCS or W)

measurements without calibration

standards. It operates with uniform

low fi eld conditions, allowing the drift

time information for ions to be used

to determine collision cross section

measurements. With the Agilent

exclusive iFunnel technology, this

instrument dramatically increases

the ion sampling into the mass

spectrometer, and results in higher

quality MS/MS spectra at trace levels.

For more details on this technology,

read Agilent Technical Overview

5991-3244EN. Realize signifi cant gains in ion mobility performanceThe 6560 delivers an optimized uniform

drift fi eld mobility cell and interface to

a high resolution Q-TOF instrument,

providing a signifi cant gain in ion

mobility performance. The use of ion

funnel technology pioneered by Agilent

for both triple quadrupole and Q-TOF

instruments over the past 3 years has

been incorporated into the IM-QTOF

system. This has resulted in combined

ion mobility separation and mass

resolution with high sensitivity.

On the following pages, we’ll share

applications from collaborators using

the Agilent 6560 for a variety of

analyses. The examples are grouped

into four categories, as discussed, to

demonstrate the key capabilities of the

system:

1. Resolving structural isomers

2. Increased peak capacity and

specifi city

3. Find and confi rm minor

components

4. Preserve protein conformations

4

Resolve Structural IsomersApplication examples

Aldicarb-sulfone (C7H

14N

2O

4S)

[M+Na]+ = 245.056649

Acetamiprid (C10

H11

ClN4)

[M+Na]+ = 245.056445

DMass = 0.2 mDa, which requires ~ 2,000,000 resolution

Separation of isobaric pesticides

N

Cl

C

CH3

CH3N

N NH

O

O

O

NN

SO

A

Theoretical plot

No

rmalize

d a

bu

ndan

ce %

245.055 245.056 245.057 245.0580

0.2

0.4

0.6

0.8

1.0

Mass-to-charge (m/z)

(1:10)

No

rmalize

d a

bu

ndan

ce %

245.055 245.056 245.057 245.0580

0.2

0.4

0.6

0.8

1.0


B

×106

0

0.2

0.4

0.6

0.8

118.297

Drift time (ms)Drift time (ms)

17 18 19 20 21

Aldicarb-sulfone

×104

0

1

2

3

4 19.441

17 18 19 20 21

Acetamiprid

IMS Drift

separation

C

×104

Drift time (ms)

17 18 19 20 21

1

0

2

3

4

5

m/z 245.013827–245.177238

RT = 0.026–1.987 minutes

*18.297

*19.441

D

This example shows the combined separation power of UHPLC, ion mobility, and mass resolution. Two pesticides, differing in

mass by less than 0.2 millidaltons, require overall separation power of approximately 2,000,000x to resolve them. B) shows the

theoretical plot of the two compounds. C) and D) show clear IMS drift separation of the two compounds (blue and red), which

are separated by 1.144 milliseconds in drift time. Even at a concentration difference of 10:1 between the two compounds, the

drift resolution is suffi cient to separate the two isobaric compounds without the use of UHPLC.

5

HOOH

OH

OHO O

O

HOOH

OH

OHO O

O

Citrate (C6H

8O

7)

Isoitrate (C6H

8O

7)

Counts

Drif

t tim

e (m

s)

190.8 190.9 191.0 191.1 191.2 191.3 191.4 191.50

0.20.40.60.81.01.21.4

×104

Coun

ts


Mass spectrum

10,000

13

14

1515.02

15.74

17.43

19.66

16

17

18

19

20

Drif

t tim

e (m

s)

Drift spectrum

190.7 190.8 190.9 191.0 191.1 191.2 191.3 191.4 191.5 191.6

13

14

15

16

17

18

19

20


14 15

15.02

15.74

17.43

16 17 180

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8×104

Drift time (ms)

Coun

ts

Drift spectrum

Separation of isomers: citrate and isocitrate

Resolve Structural Isomers Examples

Citrate and isocitrate are isomers that are very similar in physical properties and structure. This presents challenges in their

bio-analytical characterization. The Agilent 6560 Ion Mobility Q-TOF LC/MS shows clear resolution between these isomers.

6

HO OH

O OHO

OHO

O

Separation of methylmalonic acid (MMA) and succinic acid (SA)

MMA (C4H

6O

4) SA (C

4H

6O

4)

MMA

MMA(dimer)

SA(dimer)

SA

Counts

Drif

t tim

e (m

s)

100 102 104 106 108 110 112 114 116 118

117.0191

114.9015

120 122 124 126 128 1300

0.10.20.30.40.50.60.70.80.91.0

×106

Coun

ts


MMA and SA are isomersthat cannot be separated by MS

MMA and SA can be separated by IMS

400,000 200,000

17.4

17.6

17.8

18.0

18.2

18.4

18.6

18.8

19.0

19.2

19.4

19.6

18.14

18.86

Drif

t tim

e (m

s)

Drift spectrum

Mass spectrum

116.8 116.9 117.0 117.1 117.2 117.3

17.5

18.0

18.5

19.0

19.5


This is another example showing the ion mobility separation of the isomers of MMA, which is the vitamin B12 defi ciency

marker. In human plasma, its isomer (SA) is approximately 20 to 100x higher in concentration than MMA. These isomers are

very diffi cult to separate by HPLC due to their polar properties. Our data demonstrate that the 6560 can resolve these isomers

clearly, and the result can be used for accurate quantitation.


7

Resolving structural sugar isomers C18H32O16

CountsD

rift t

ime

(ms)

Mass-to-charge (m/z)400,000 200,000

24.624.825.025.225.425.625.826.026.226.426.626.827.027.227.427.6

25.77

26.69

Drif

t tim

e (m

s)

Drift spectrum

526 527 528 529 530

17.5

18.0

18.5

19.0

19.5

526.0 526.5 527.0 527.5 528.0 528.5 529.0 529.5 530.0 530.5

528.1607

527.1580

0

2

4

×104Co

unts

Mass spectrum


Melezitose

Resolving two isobaric tri-saccharidesRaffinose

H

HH

HOH

HO

HO

HO

H

O

O

H

HHO

HH

HO

HO

H

O

O

OH

OH

OH

OH

H

H

H

O

H

H

H HOH

OH

OH

OH

HO

HO

HO

H

O

H

H

HOH

H HO

O

OH

OH

OH

H

HH

O

O

This example demonstrates how ion mobility can be used to resolve two different isobaric tri-saccharides with the same

exact mass. These isomeric structural differences cannot be resolved using an traditional premium high resolution mass

spectrometry system.


8

Silymarin, the active extract from plants, contains a mixture of fl avonolignans consisting of silibinin, isosilibinin, silicristin,

silidianin, and others. The most active compound in silymarin is silibinin. It has been extensively used in patients with liver

disease. Ion mobility can be used to characterize these silymarin isomers. In this study, various isomers of silymarin are

separated, and each of their unique collision cross-section values are determined (data not shown).


IM Q-TOF Analysis on the silymarin family

Counts

Drif

t tim

e (m

s)


[M+Na]+

Silycristin

Silydianin

Silybinin/Isosilybinin

2,000,000 1,000,000

25.0

24.5

25.5

26.0

26.5

27.0

27.5

28.0

28.5

29.0

29.5

30.0

30.5

28.14

26.56

25.22

29.41

Drif

t tim

e (m

s)

Drift spectrum

502 504 506 508 510 512

25

26

27

28

29

30

502 503 504 505 506 507 508 509 510 511

506.1093

507.1118

505.1059

0.5

1.0

1.5

2.0

×106

Coun

ts

Mass spectrum


Silibinin

HO

OHOH

OH

AB

2’3’

O

OCH

CH2OH

O

O

O

Isosilibinin

HO

OHOH

OHA

B

O

OCH3

CH2OH

O

O

O

Silchristin

HO

OHOH

OH

OH

O

OCH3

CH2OH

O

O

Silidianin

HO

HO

HOO

O

HO OCH3

H

OH

O

O

H

9

30

40

50

60

Ion m

oblity

dri

ft t

ime (

ms)

Carbohydrates analysis by IM-MS

20

10

0

0 500 1,000

Mass (Da)

1,018 1,020 1,022 1,024 1,026 1,028

Mass (Da)

35

3

36 37 38 39 40 41 42

Drift time (ms)

Lacto-N-difucohexaose II

Mixture of lacto-N-difucohexaose I and II

Lacto-N-difucohexaose I

Oligosaccharide mixture

(Profs. John McLean and Jody May, Vanderbilt Univ.)

Fuc

Gal Gal GlcGlcNAc

Fuc

Fuc

Gal Gal GlcGlcNAc

Fuc

Lacto-N-difucohexaose I

Lacto-N-difucohexaose II

A mixture of lacto-N-difucohexaose I/II isomers, which differ by the location of the fuctose group, is discovered in a complex

sample mixture using the 6560 IM-Q-TOF. Upon individually analyzing the standards, the observation of two species of human

milk oligosaccharide isomers is confi rmed.


10

GC-APCI/IMS-Q-TOF analysis of ASTM compound mixture

×107

0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

RT = 4.3 min

4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0

0.40.60.81.01.21.4

Acquisition time (min)

Counts

×105

113 114 115 116 117

115.0567

116.0592 117.0708 120.0902

118 119 120 121 122 123 1240

2

Counts


Drift time (ms)113 114 115 116 117 118 119 120 121 122 123 124

8

9

10

11

Mass

-to-c

harg

e (

m/z

)

Mass spectrum

TIC

Counts2,000,000 1,000,000

8.08.48.89.29.6

10.010.410.811.211.6

10.34

9.28

8.96

9.93

110.2

Drif

t tim

e (m

s)

Drift spectrum

Some possibleisomer structures

CH3

CH3

CH3

CH3

CH3

CH3

H3C

119.0891

In this study, we use the GC-APCI interface coupled with ion mobility to analyze the ASTM standard compound mixture. For

example, at the GC peak of 4.3 minutes, more than three separated compounds with many associated isomers in the mass

range of 110 to 125 Da were found. Therefore, the Agilent 6560 provides higher peak capacity to successfully resolve the

different isomers.


11

Moblity

dri

ft t

ime (

ms)

Ion mobility provides greater specificity

100


Crude bacterial extract


1,444 1,446 1,448 1,450 1,452 1,454


Integrated mass spectrum

Mobility-filtered mass spectrum

S/N increases significantly

20

30

40

50

500 1,000 1,500 2,000

1,444 1,446 1,448 1,450 1,452 1,454


Increased Peak Capacity/Specifi cityApplication examples

Another value of ion mobility is to effectively clean up background chemical noise from a crude bacterial extract. This mobility

heat map shows hundreds of components in the sample with overlapping compounds at nearly every m/z value. In the

highlighted polygon region of the heat map, see the integrated mass spectrum that has too many ions to provide confi dent

compound identifi cation. The bottom graphic shows the mobility-fi ltered mass spectrum, which eliminates many of the

overlapping chemical background ions. This enables fast and confi dent compound identifi cation.

12

Counts

Ion mobility provides greater specificity

1

0.4

RNAseB Native glycans anaysis Enable the extraction of ion series of interest - a group of glycans from matrix for further study. (Prof. Cathy Costello, Boston University)

2

3

4

5

6

7

0.8 1.2 1.6 2.0 2.4 2.8 3.2 3.6 4.0 4.4 4.8 5.2 5.6 6.0 6.4 6.8 7.2 7.6 8.0 8.4 8.8 9.2 9.6 10.0

×105D

rift

tim

e (

ms)


100

15

20

25

30

35

40

500 1,000 1,500 2,000


In this example, Professor Cathy Costello at Boston University uses an Agilent 6560 Ion Mobility Q-TOF LC/MS system to

selectively isolate a group of RNaseB glycopeptides from the background matrix. A selected region from an LC chromatogram

(blue) is displayed in the IMS-MS heat map (insert). One region of distinct mass differences (white box), as shown by

the trend line, is clearly distinguishable as a trendline with increasing number of monosaccharides in the glycopeptide

compositions. The signal from these glycopeptide ions can be selected in the mobility trace and separated from other ions

present in the spectrum, for further study.

Increased Peak Capacity/Specifi city Examples

13

Ion mobility simplifies complex spectraRNAseB glycopeptides NLTK- Man5 through 9

Unfiltered mass spectrum

Mobility-filtered mass spectrum

B

A×103

×102

0

0.1

0.2

487.2

006

846.3

583

927.3

846

1008.4

113

1089.4

374

1179.9

666

5

2

2

2

2

2

635.0

031

846.3

588

927.3

846

1008.4

113 1089.4

374

1179.9

6664

2

2

2

2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

Counts

0

1

2

3

4

5

6

7

8

9

Counts


200 400 600 800 1,000 1,200 1,400 1,600

NLT

K-

Man

5

NLT

K-

Man

6

NLT

K-

Man7

NLT

K-

Man

8

NLT

K-

Man

9

1,800 2,000 2,200 2,400 2,600 2,800 3,000

In this example, ion mobility is used to simplify complex glycopeptide spectra. A) RNaseB glycopeptides on a custom

HILIC-C18 HPLC-chip on the 6560 shows all of the combined ions corresponding to compounds eluting in the selected

retention time window. B) RNaseB glycopeptides on HILIC-C18 chip on the 6560 with IMS separation shows a simplifi ed

mobility spectrum for the different glycoforms of a single glycopeptide. The peaks with a distinct delta of 162 correspond to

increasing number of mannose units in the high-mannose N-linked glycopeptide NLTK, as labeled.


14

Ion m

obilit

y dri

ft t

ime (

ms)

Mass-to-charge (m/z)0

0

20

40

50

500 1,000 1,500 2,000

10

30

60

70

PE 35: (6-2) PE 37: (8-4) PE 39: (10-6)PE 33: (4-2)

+Na +K

740 760 770 780 790750 800 810 820

PE 60:NPE 62:N

PE 64:N

PE 33:N

PE 35:N

PE 37:N

PE 39:N

PE 41:N

PE 2 3:N

PE 21:N

PE 19:N

PE oligomers +2 lipids

+3 lipids

+1 lipids

+4 lipids

Lipid analysis: Mixture of L-a-phosphatidylethanolamine (PE) lipids

Structure of predominant species

HO

O

O

O

NH3

+

PO O O


In this example, a class of lipids, the phosphatidylethanolamines, is separated by ion mobility. Professor John McLean’s group

at Vanderbilt University quickly identifi es over 200 different lipids and oligomers that fall on a specifi c trend line. Another

interesting discovery is made while evaluating the data within the main trend of the lipids; they observe a secondary trend

consisting of differing degrees of unsaturation.


15

Ion mobility of polymeric ink dispersants

Counts

Dri

ft t

ime (

ms)

Mass-to-charge (m/z)15,000,000 5,000,000

24

26

28

30

32

34

36

38

40

42

44

46

48

50

Drif

t tim

e (m

s)

Drift spectrum

TIC

500 600

1

700 800

+4

+3

+1

+2

Zoom

900 1,000 1,100 1,200 1,300 1,400

25

30

35

40

45

50

Counts

Acquisition time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

1

3

5

7

450 550 650 750 850 950 1,050 1,150 1,250 1,350 1,450

676.4525

1,221.9958

0

0.5

1.0

1.5

×106

×107

Counts

Mass spectrum


In this example, ion mobility is used to identify different hydrocarbon ion series in polymeric ink dispersants. The trend-lines

illustrating the various charge-state hydrocarbon ion series are clearly resolved. In addition, the ion mobility specifi city can

signifi cantly reduce the background matrix effect.


16

Ion mobility of polymeric ink dispersants – hydrocarbon molecules

Drif

t tim

e (m

s)


938 939 940 941 942 943

+1

+3

+2

32

34

36

38

40

Coun

ts

Massspectrum

938.0 938.4 938.8

938.6302939.1318

939.6333

940.1348939.9505

940.2875940.6197

940.9549

941.2875941.6221

940.8207

941.1309 942.1170 942.6231

939.2 939.6 940.0 940.4 940.8 941.2 941.6 942.0 942.4 942.80

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8×105


Resolving overlapping charge-state isotopes by ion mobility

In this example, ion mobility is used to resolve overlapping charge-state isotopes that were unresolved by mass resolution

only. This information is used to compare and confi rm the quality level of the various ink products.


17

Ion mobility of diesel (hydrocarbons) sample

Background is noise reduced significantly

Enable the extraction of ion series

of interest for further study

200 400 600 800 1,000 1,200 1,400 1,600 1,800 2,000 2,200 2,400 2,600 2,800 3,000

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

500 1,000 1,500 2,000 2,500 3,000

15

20

25

30

35

×106

Dri

ft t

ime (

ms)



Counts

Mass spectrum

200 400 600 800 1,000

937.1869 1,153.2286

1,200 1,400 1,600 1,800 2,000 2,200 2,400 2,600 2,800 3,0000

1

2

3

4

5

×105

Mass spectrum

In this example, ion mobility is used to identify different hydrocarbon ion series in a diesel sample. The middle panel shows

how ion mobility enables the extraction of ion series of interest for further study. The lower panel shows how ion mobility

signifi cantly reduces the background noise.


18

In this example, ion mobility is used to enhance the identifi cation of tryptic peptides in mouse (A) and human blood plasma,

which is useful for analyzing disease states. The inset graphic (B) shows a zoomed-in region of the 3-D plot where 10 peptides

were identifi ed easily with IMS in 0.5 seconds of the 15-minute LC run (red circles). By comparison, the same sample was run

with a 100-minute LC gradient, using a high-resolution MS instrument, which yields only three identifi cations at the same

region (indicated by red asterisks). In summary, the 6560 detects > 3x the peptides > 5x faster.

Ion mobility provides greater detection for proteomicsProtein digest, Dr. Erin Baker, PNNL

1,330,000A B

0

0

250

400

m/z

700

850

17.5 22.5 37.5Drift time (ms) 42.5

543.7

5545.5

0m

/z549.0

0550.7

5

22.7 23.4 25.5Drift time (ms) 26.2

2,430,000

49,400

0

026,600

Find and Confi rm Minor ComponentsApplication examples

19

IMS-MS for discovery proteomics: transmembrane spanning peptides of HeLa digest

High dt

High dt

All ionsfragmentation

Low dt

Low dt

Low dt

200 400 600 800 1,000 1,200 1,400 1,60015

20

25

30

Drif

t tim

e (m

s)


200 400 600

86.0964 300.1545

526.3220

734.8716

892.9430

800 1,000 1,200 1,4000

1

2

3

4

5

Coun

ts


346 350 354 3580

20

40

60

80

100

110

120

140

160

Inte

nsity

Retention time

×103

345 350 355 3600

10

20

30

40

50

60

70

Inte

nsity

Retention time

×103

In this sample, we are able to identify multiple potential isoforms of transmembrane-spanning peptides using ion mobility.

Using Agilent All Ions MS/MS and an MS/MS library from our Spectrum Mill proteomics software, we use Skyline software

from the MacCoss Group at the University of Washington to match some transmembrane-spanning peptides with known

helical structures. In this way, we are able to determine the relative quantitation ratio between an helical form (condensed,

lower dt) and a denatured form (higher dt).

Find and Confi rm Minor Components Examples

20

Sensitivity: detection limit of spiked compound in urine

Q-TOF mode IM mode

1 nM

10 nM

50 nM

100 nM

1,000 nM

10,000 nM

0.5

1.0

1.5

×104

0.40.81.2

×104

0.5

1.5

2.5×104

1234

123

×104

×105

0.5

1.5

2.5

×106


Coun

tsCo

unts

Coun

tsCo

unts

Coun

tsCo

unts

3.8 4.0 4.2 4.4 4.6 4.8 5.0

0.20.40.60.8

246

×101

0.5

1.0

1.5

0.5

1.5

2.5

0.5

1.5

2.5

×102

×102

×103

0.5

1.5

2.5×104


Coun

tsCo

unts

Coun

tsCo

unts

Coun

tsCo

unts

4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8

This example shows a comparison of detection limits between the Q-TOF and the IM Q-TOF modes of the system for a

spiked compound (biological marker) in urine. Although very similar limit of detection (LOD) sensitivity is observed between

these data acquisition modes, superior signal-to-noise (S/N) data using the IM-Q-TOF mode are obtained. Improvement in

quantitative results are achieved due to the much lower background noise level.

Find and Confi rm Minor Components Examples

21

IM Comparison on Cytochrom C (+8): (uniform drift tube versus travelling wave)

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

0.20.40.60.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.6

Coun

ts

%

Drift time (ms)

Drift time (ms)

Drift spectrumTravelling wave IM data

Preserve protein native structures better due to the much lower ions heating effects

S1: NativeS2–S5: Denatured

S3

S1

S2

S4

S5

RF 90 V

Neutral condition

Denatured condition

RF 150 V

16.43

25.01

6.16

19.18

22.72 26.0028.45

32.24 36.55

×107

7 8 9 10

9.79

?

10.35

7.86

11 12 136543210

100

The minimal ion heating effect from the Agilent 6560 Ion Mobility Q-TOF LC/MS system is critical for maintaining a native

protein conformation. By altering the trap RF voltages, we can change the protein conformation from its native state (S1) to

various degrees of denatured states (S2–S5). By comparison with an alternative travelling wave IM system, the Agilent drift

tube IM system requires a much lower energy, minimizing the ion heating effect that causes the denaturation of the protein

molecules.

Preserve Protein Conformations Examples

22

Ion mobility Q-TOF on protein isoform analysis

1,000 1,100 1,200 1,300 1,400 1,500 1,600 1,70020

25

30

35

40

Drif

t tim

e (m

s)


x104

x105

x104

x104

0.50

11.5

22.5

33.5

44.5

55.5

1,348.0819

1,497.7578

1,225.6216842.8653793.4033

963.2035

1,123.4858

1,037.2184

1,684.8532

710.0444

650 750 850 950 1050 1150 1250 1350 1450 1550 1650 1750

0

0.5

1

1.5

21,348.0815

1,225.6208

1,123.5678 1,497.75791,037.2166

1,050 1,150 1,250 1,350 1,450 1,550 1,650

0

2

4

6

81,497.7583

1,348.08251,684.8544

1,225.5310

1,123.5693

0

2

4

6 1,348.0835 1,497.6459

1,225.5305

1,123.48851,684.7309

Cry34AB

Counts

Counts

Counts

Counts



1,050 1,150 1,250 1,350 1,450 1,550 1,650Mass-to-charge (m/z)

1,050 1,150 1,250 1,350 1,450 1,550 1,650Mass-to-charge (m/z)

A protein sample (CRY34AB) under native and denaturating conditions is analyzed using ion mobility. Using just the IM

information, it appears that there are possibly three isoforms, which are clearly separated. The IM results also confi rm that

the different isoforms generate different charge envelopes, indicating various protein folding structures, consistent with

time-consuming X-ray crystallography.


23

Resolving isoforms of IgG-2

2,000 2,200 2,400 2,600 2,800 3,000

20

25

30

Drif

t tim

e (m

s)


Only one conformation detected

Denaturated condition

x105

0.5

0

1

1.5

2

2.5

A B

1,850 1,950 2,050 2,150 2,250

69+

66+

63+

59+56+

53+

50+

2,350 2,450 2,550 2,650 2,750 2,850 2,950 3,050

Mass spectrum

mAb Structure

Publication Paul Schnier

IgG2-A isoform IgG2-B isoform

Counts


The IgG-2 molecule has two different conformations (isoforms A and B) under native conditions. However, normal LC/MS

conditions, with a high content of organic solvent and 0.1% formic acid, will destroy the native structure. Only one denaturated

protein conformation can then be detected.


24

IM-Q-TOF Analysis of native IgG-2

5,000 5,500 6,000 6,500 7,000

35

40

45

50

55

Drif

t tim

e (m

s)


Two possible conformations detected

A

B

x103

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4,800 5,000 5,200 5,400 5,600 5,800 6,000 6,200 6,400 6,600 6,800 7,000 7,200

30+

29+

28+

27+

26+

25+

24+

23+

22+21+

Mass spectrum

Counts


Under native conditions (100 mM ammonium acetate), the IgG-2 charge envelope will shift to higher m/z (5,000–7,000 range).

Two protein conformations were clearly detected. Isoform A is the true native structure, and isoform B represents the IgG-2

with possible mismatch in disulfi de bonds.


25

IM-Q-TOF Analysis of native IgG-1

5,000 5,500 6,000 6,500 7,000

35

30

40

45

50

55

Drif

t tim

e (m

s)


Two possible conformations detected

A

B

x104

0.5

1.0

1.5

2.0

5,000 5,200 5,400 5,600 5,800 6,000 6,200 6,400 6,600 6,800 7,000

30+

29+

28+

27+

26+

25+

24+

23+22+ 21+

Mass spectrum

Counts


Two protein conformations are detected in the native IgG-1 sample using the 6560 system.


26

IM-Q-TOF Comparison of IgG-1 and IgG-2

5,000 5,500 6,000 6,500 7,000

Drif

t tim

e (m

s)


IgG-2 (22+ charge state) has more of the B form

IgG-2 22+

22+

5,000 5,500 6,000 6,500 7,000

32 34 36 38 40 42 44 46 48 50 52 54 56 58

35

40

45

50

55

35

40

45

50

55

Drif

t tim

e (m

s)


IgG-1

0.2 38.60

43.81

48.16

55.59

0.4

0.6

0.8

1.0

1.2

1.4

×105

Coun

tsCo

unts

IgG-1

(22+)

1

42.14

Drift spectrum

Drift spectrum

46.06

49.43

54.90

2

3

4

5

6

7

×104

Drift time (ms)

32 34 36 38 40 42 44 46 48 50 52 54 56 58Drift time (ms)

IgG-2

(22+)

A

A

B

B

In this example, ion mobility is used to determine the relative amount of isoforms A and B in the IgG-1 and IgG-2 sample.

In a side-by-side comparison, IgG-1 posts a slightly higher percentage of isoform A (native) than B at its 22+ charge state

molecule. Conversely, a higher percentage of isoform B is detected in the IgG-2 sample.


27

Ion Mobility Adds A New Dimension to Your ResearchThe Agilent 6560 Ion Mobility Q-TOF

LC/MS is the fi rst commercial

instrument that enables researchers to

address truly fundamental questions

about structure, function, and the

workings of complex biological

systems with real confi dence and

ease. Combining the orthogonal

separation techniques of liquid

chromatography, mass measurement

and ion mobility tremendously

increases peak capacity, giving you the

ability to more effectively characterize

a variety of molecules.

The technology in the 6560 IM

Q-TOF provide important benefi ts for

analytical challenges:

• Added separation capability for

isomeric compounds

• Increased peak capacity/specifi city

• Added selectivity for very complex

samples

• Reduced effect of chemical

backgrounds

• Ability to distinguish different

protein conformations

Simply put, you can now resolve and

detect a greater number of compounds

and components than ever before.

Acknowledgments• Cathy Costello, Ph.D.,

Boston University

• Joseph Zaia, Ph.D.,

Boston University

• Rebecca S. Glaskin, Ph.D.,

Boston University

• Kshitij Khatri,

Boston University

• John McLean, Ph.D.,

Vanderbilt University

• Jody May, Ph.D.,


• Cody Goodwin, Ph.D.,


• Erin Baker, Ph.D.,

Pacifi c Northwest National Labs

• Agilent Technologies Scientists:

Huy Bui, PhD.

Crystal Cody, Ph.D.

Ed Darland, Ph.D,

John Fjeldsted, Ph.D.

Chris Klein, Ph.D.

Ruwan Kurulugama, Ph.D.

Sheher Mohsin, Ph.D.

Alex Mordehai, Ph.D.

Gregor Overney, PhD.

George Stafford, Ph.D.

Joachim Thiemann, Ph.D.

Bruce Wang

For research use only. Not for use in diagnostic

procedures.

This information is subject to change without notice.

© Agilent Technologies, Inc., 2015

Published in the USA, May 29, 2015

5991-5723EN

Learn morewww.agilent.com/chem/imq-tof

Contact an Agilent Representative

agilent.com/chem/quote

U.S. and Canada

[email protected]

Europe

[email protected]

New Features of MassHunter Software with Ion Mobility Allow You To Go Deeper into Your Data:• Novel Swarm Autotune tunes the

mass spec in one quarter of the

time

• 4-D feature fi nding in IM-MS

Browser

• Single fi eld CCS calculation

• Differential profi ling using Mass

Profi ler, including statistical analysis

and PCA plots

Learn more at www.agilent.com/

chem/masshunter

add a new dimension to your research … measure of conﬁdence add a new dimension to your research...

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