The usefulness of Ion Mobility-Mass Spectrometry for Small Molecules Analysis

The usefulness of Ion Mobility-Mass

Spectrometry for Small Molecules

Analysis

J.  Far

a

_{,  S.  Goscinny}

b

_{,  L.  Joly}

a,b

_{,  R.  Touilloux}

a

_{,  J.  Echterbille}

a

_{,  L.  Quinton}

a

 _{,  G.  Eppe}

a

 _{and  E.  De  Pauw}

a  

a  

_{Laboratory  of  Mass  Spectrometry,  University  of  Liege,  3  allée  de  la  chimie,  Bat  B6C  4000  Liege}  

b  

_{ScienNfic  InsNtute  of  Public  Health,  PesNcide  Unit,  JulieRe  Wytsman  14,  BE-­‐1050  Brussels,  Belgium}  

• 

Breve  introducNon  to  Ion-­‐mobility  Spectroscopy  

• 

IMS  for  small  molecules  (low  molecular  weight)  

– 

Metallomics  (selenometabolomics  –  proof  of  concept)  

and  de-­‐novo  structural  assignaNon  

– 

PesNcides  screening  (S.  Goscinny  et  al.)  

– 

Disulfide  bridge  assignaNon  in  oligopepNde  for  

venomics  (proof  of  concept;  J.  Echterbille  et  al.)  

• 

conclusions  

The Drift Tube Ion Mobility Mass Spectrometry

3

Concept of Ion Mobility

Ion Mobility Spectrometry is usually the technique of choice of

(bio)macromolecules like proteins and DNA

•  G. Gabellica

et al.

, G-quadruplex structure determination

V. Gabellica

et al.

, J. Am. Chem. Soc., 2007,

129

: 895-904

•  Proteins structures analysis (e.g. prions)

Hilton

et al.

, J. Mass Spectrom, 2010,

21 (5):

845-854

Illustration from The Bowers group website:

•  DTIMS (previous slide)

Drift-Time Ion Mobility Spectrometry

•  AIMS

aspiration ion mobility spectrometry

Zimmermann and coworkers, Sensors and Actuators B,

2007: 428-434

•  DMS / FAIMS

Differential-Mobility Spectrometry

Field-Asymmetric waveform Ion Mobility Spectrometry

Kolakowski and Mester, Analyst, 2007 132: 842-854

•  TWIMS (next slide)

•  General principles of Travelling Wave Ion Mobility Spectroscopy:

Ion Mobility Mass Spectrometry

Waters Synapt G2 HDMS

Courtesie  of  

Waters  

5

!

=

(​

3

$/

16

& )​(​

2

)/*+ )↑​

1

⁄

2

  ​(​.

+

//./ )↑​

1

⁄

2

  (​

1

/

Ω

 )

• 

K: ion mobility constant

q: charge of ion (Coulomb)

• 

N: density of buffer gas

k: Boltzmann’s constant

• 

T: temperature (Kelvin)

• 

m: mass of gas, M mass of ion

→ reduced mass

≈

m

_gas

m/z

165

170

175

180

185

190

195

200

205

210

215

%

0

100

09-05-2011_SEMET 10PPM IM-MS MODE POS HR 36 (3.719) Cm (2:200)

6.49e4

180.9748 178.9755 176.9785 174.9899 198.0030 182.9763 196.0034 193.0002 200.0004

•  ESI MS SeMet and reflectron in V optic mode:

• 

Δm = -2.02 ppm,

•  R

_FWHM

≈ 12 000

•  Loss of 17,0282 (NH

₃

?)

(198.0030-180.9748)

•  NH3 = 17.0265

– 

Δm = 98.8 ppm !!!

•  ESI MS SeMet and reflectron in W optic mode :

• 

Δm SeMet = -2.02 ppm, R

_FWHM

≈ 25 000

m/z 195.940 195.980 196.020 % 0 100

196.0034

195.9881

m/z 193.940 193.980 194.020 % 0 100

194.0048

193.9895

m/z 191.950 192.000 192.050 % 0 100

191.9891

191.9072

192.0103

192.0255

192.0770

J.  Far,  G.  Mazzucchelli,  E.  De  Pauw  ,  G.  Eppe  

Structural assignation of an isobar compound in a

Scan

20

40

60

80

100

120

140

160

180

200

%

0

100

SeMet 10ppm microESI IM-MS MSe N2 HR_dt_01

TIC

5.11e4

3.71 2.76 4.35 8.80 5.51 10.18 12.19

Structural assignation of an isobar compound

in a L or D,L- SeMet standard by ESI IM MS

m/z

165

170

175

180

185

190

195

200

205

210

215

%

0

100

09-05-2011_SEMET 10PPM IM-MS MODE POS HR 42 (4.356)

7.85e3

198.0030 196.0034 194.0048 _200.0035

m/z

165

170

175

180

185

190

195

200

205

210

215

%

0

100

09-05-2011_SEMET 10PPM IM-MS MODE POS HR 38 (3.931)

5.40e3

180.9748 178.9755 176.9785 182.9763 195.9850 193.9895

7

Se

N

H

₃+

O

O

H

O

O

H

Se

+

N

H

C

H

₃

J.  Far,  G.  Mazzucchelli,  E.  De  Pauw  ,  G.  Eppe  

0.00

2.50

5.00

%

100

3.93

7.50

XIC 196

N

-2,3-dihydroxypropionyl-selenocystathionine

•

 

Major selenocompound

in the low molecular weight water extract from

Se-rich yeast

•  [C

₁₀

H

₁₈

N

₂

O

₇

Se + H

+

_]

_m/z

_{: 359.0358}

•  Present in various batches of commercial Se-rich yeast [Gil-Casal and

coworkers Metallomics (2010)] and Lab-made Se-rich yeast [Rao and

coworker Anal. Chem. (2010)]

O

H

N

H

SeH

+

O

OH

OH

O

N

H

₃ +

OH

O

Se

N

H

₃ +

OH

O

m/z:  181.9720  

m/z

:  176.0559  

N

H

₂ +

O

OH

O

H

O

OH

m/z:  88.0399  

m/z

:  269.9881  

O

H

N

H

O

O

OH

OH

Se

N

H

₃ +

OH

O

O

H

N

H

O

OH

OH

Se

N

H

₃ +

OH

O

O

m/z:  359.0358  

Specific  fragments  

•  Metabolic origin or by-product

? (No sulfur analogue describe in the literature)

•

 

Two coeluted isomers

, confirmation on the basis of (MS

2

), MS

3

and MS

4

data

2D-LC purification with of selenometabolites using

ICP/MS and ESI ToF MS(/MS) detection

•  Purification of m/z: 359 according to Dernovics and coworkers,

(2009) metallomics, with modifications

ESI  ToF  MS(/MS)  

m/z 50 100 150 200 250 300 350 400 450 500 550 % 0 100 176.0970 130.0526 84.0459 197.0574 282.2885 229.1525 489.1249 371.1152 511.1140 m/z 350 352 354 356 358 360 362 % 0 100 353.0215 351.0772 356.0181 360.1632 _360.9686 363.0983 359.0439 357.0418 355.0453 354.0903 362.0659

9

S.

A

.X

2nd _{LC dimension: Anion Exchange HPLC}

(PRPX-100) 25 10 t o 25 0mM C H3 CO 2 NH 4 pH 5.5 50 75 0 1 2 3 4 10 20 30 40 50 60 70 si gn al ( cp s) x 10 +4

Elution time (minutes)

Se82 Se78 m/z:  661   m/z:  604   m/z:  370   m/z:  313   and  373   m/z:  345   ???   To  idenNfy   m/z:  400  

J.  Far,  E.  De  Pauw  Edwin,  R.  Lobinski,  G.  Eppe  

50 100 150 200 250 Superdex  75   LxID:400x30mm   0 1 2 3 4 5 6 7 8 9 si gn al (x 10 +5 cp s) time (minutes)

First LC dimension: Size Exclusion Chromatography

82_Se 78_Se

ICP Q MS

Pr

ep

ar

ati

ve

S.

E.

C

100

 

200

 

300

 

400

 

500

 

600

 

700

 

800

 

900

 

1000

 

m/z

 

0

 

350

 

700

 

741

 

In te n si ty , c o u n ts

 

276.1033

 

130.0378

 

359.0161

 

489.0836

 

% 0 100

359.0412

357.0391 338.9983 332.9796 324.0644 286.9926 247.1014 176.0898 381.0240 383.0237 388.2612449.6294 50 100 150 200 250 300 350 400 450 500 550 m/z

3D-­‐LC  ESI  QToF  MS  

Dernovics  and  coworkers,  Metallomics  (2009)  1,  317–239  

2D-­‐LC  ESI  IMS  ToF  MS  

This  work  

Clean-up of mass spectra by IM-MS:

Scan

20

40

60

80

100

120

140

160

180

200

%

0

100

m/z

356 357 358 359 360

%

0

100

100 cps

358.0454

357.0120

359.0472

359.2626

360.0089

%

0

100

157 cps

_359.2792

359.0472

358.3516

357.3095

360.2827

356 357 358 359 360

m/z

 ≈ 12%  

Selection of m/z: 357 and 359 on quad, then IMS and MS spectra

Separation of

N

-2,3-DiHydroxyPropionyl-SelenoCystathionine by

ESI IM-MS and estimation of isomer ratio

m/z 50 75 100 125 150 175 200 225 250 275 300 325 350 % 0 100 135.9715 266 133.9702 84.0826 129.1065 164.9348 181.9741 359.0430 260.2022 222.0497 280.0170 357.0492 MS/MS 359 bin 138 269.9879 Noisy m/z % 0 100 176.0601 152 118.9706 56.0494 103.0424 70.0642 168.0262 255.0025 249.0403 181.9741 248.9506 269.9900 358.0413 327.2386 296.8937 MS/MS 359 bin 82 176.0543 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 m/z 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 % 0 100 181.9741 711 179.9684 56.0494 158.0457 132.0592 88.0369 359.0347 357.0410 271.0183 269.9900 183.9908 339.1695 297.2027 MS/MS 359 bin 80-140

Confirmation

experiments in

progress

0 50 100 150 200 250 300 350 400 50 60 70 80 90 100 110 120 130 140 150

XIC  IMS  

m/z:  176,0

m/z:  269,9

Dernovics  and  Lobinski,  Anal.  

Chem,  (2008)  80:  3975-­‐3984  

SEC/SAX/HILIC-­‐

ESI  FT  Orbitrap  

MS  

Ion Mobility Mass Spectrometry for

pesticides

screening

•  Pesticides are rich in diversity:

–  chemical structure,

–  solubility, volatility,

–  Persistence in organisms and environment

•  in number: More than 1200 molecules

–  around 740 are allowed to be used in the EU

–  around 500 compounds sought/sample

•  LC based separation is the major used analytical

method

•  Pesticides screening: Multiresidues methods are

mandatory

S. Goscinny, L. Joly, E. De Pauw,

V. Hanot and G. Eppe

Plackett-Burman design

•  5 parameters

–  IMS T-Wave velocity (m.s

-1

₎

–  IMS T-Wave Height (V)

–  Gas Pressure (mbar)

–  Helium Cell Pressure (mbar)

–  Biais (V)

•  3 responses

–  Intensity (cps, aera)

–  Resolution

–  Relative drift time (ms)

construction of the design

is done with 15 runs

4 representative

groups of pesticides

0,40

1,40

2,40

3,40

100 125 150 175 200 225 250 275 300 325 350 375 400 425 450

Dri

4

 Ti

me  (

ms

)

m/z

Carbamate

Nitrogen  pesNcide

Organophosphate

Pyrethroid

S. Goscinny, L. Joly, E. De Pauw,

V. Hanot and G. Eppe

IM-MS analyses of pesticides:

spectrum clean-up

•  Pesticides screening:

–  solubility, volatility: LC retention time

–  chemical structure: m/z in MS

–  Discrimination using drift time of isobar and coeluted compounds

m/z 200 400 600 800 1000 % 0 100 709.4479 687.4252 207.1463 134.0958 347.0227 349.2007 _473.3018 615.7142 709.9487 710.4498 711.9188 719.9089 720.9063 m/z 200 400 600 800 1000 % 0 100 207.1563 195.1463 208.1770 233.0323 235.0322 261.1438

S. Goscinny, L. Joly, E. De Pauw, V.

Hanot and G. Eppe

Water / Methanol extraction

RP-UPLC ESI MS screening of Diuron

RT: 6.38 min

[M+H]

+

_:

_233.0248

Discrimination of co-eluted molecules by

IM-MS during pesticides screening

S. Goscinny, L. Joly, E. De Pauw,

V. Hanot and G. Eppe

Pesticide:

m/z

drift time (ms)

Quinalphos

[M+H]

+

_{: 299.0619}

_3,81

Quinalphos

[M+Na]

+

_:

_321.0439

_4,97

Phenthoate

[M+H]

+

_:

_321.0384

_4,22

N

N

O

P

O

C

H

3

S

O

C

H

3

P

O

S

_O

C

H

₃

C

H

₃

S

O

O

H

3

C

Quinalphos

 

Phenthoate

 

15

•  But…

•  SS bonds imply

low fragmentation ratio

during MS/MS (CID) experiments è

Sequencing L

•  Disulfide bonds assignment

is very complex

and time-consuming!!!

Cytotoxin-­‐1  from  Naja  oxiana  venom-­‐  ~7KDa  /  5  SS  

Conotoxin  MVIIA  from  Conus  magus  -­‐  ~3KDa  /  3  SS  

IMS for peptides structure elucidation in

venomics

field

•  These bonds fulfill multiple functions :

•  Provide the toxin proper conformation to

bind to the targeted receptor

•  Reduce the immunogenicity

•  Provide a high stability in time

•  Toxins

have many disulfide brigdes

and are

highly structured

J. Echterbille, L. Quinton, E. De Pauw

Drift time (ms)

In

te

ns

ity

(a

.u

.)

Mix of native, partially

reduced and reduced forms of

a 2 disulfide bridge-containing

peptide

Four contributions to the arrival

time distribution

Mobility separation

Nano-ESI infusion

CID in the Transfer module

Recording of a total CID spectrum

Extraction

of CID

spectrum

Sequencing of each toxin’s form

Methodology: IMS for peptides structure

elucidation in venomics field

17

J. Echterbille, L. Quinton, E. De Pauw

Fragmentation spectrum of the semi-reduced forms

(arrival time : 5.371 & 5.735 ms)

 

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1100

1150

%

0

100

x7.5

x5

136.05

_299.15

186.1

1

506.28

362.16

326.65

691.41

609.85

724.43

555.28

G R

C

C

H P A

C

G K Y Y S

C

SH

SH

S

S

iY

[MH-Y]

2+

[MH-Y-Y]

2+

YY-28

PA

YS-28

[MH-P]

2+

[MH-Y-Y-S]

2+

b

2  

y

₁₂2+  

(b

6

-­‐1)

2+  

(b

₇

-­‐1)

2+  

[MH-K-Y-Y]

2+

y

₁₁2+  

[MH

3

]

3+

(y

₉

-­‐1)

2+  

[MH

3

-H

2

O]

3+

[MH

3

-H

2

O]

3+

566.32

m/z

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1100

1150

%

0

100

515.62

136.05

121.01

466.26

437.23

413.69

191.04

163.03

_208.06

299.15

712.94

669.40

587.84

555.28

614.74

G R

C

C

H P A

C

G K Y Y S

C

S

S

SH

SH

b

₁₀  

b

9  

b

₁₂2+  

b

112+  

b

133+  

b

₈2+  

b

₂  

b

102+  

b

92+  

_b

8  

b

132+  

[MH-P]

2+

(y

11

-­‐1)

2+  

y

9

-­‐1

 

y

1  

y

2  

y

₃  

_y

6  

y

₇

-­‐1

 

y

8

-­‐1

 

b

5

-­‐1  

(b

6

-­‐1)

2+  

(b

₇

-­‐1)

2+  

[MH

₃

]

3+

iY

YY-28

YS-28

[MH

3

-H

2

O]

3+

[MH

₃

-28]

3+

b

₅

-­‐1  

Time

(ms)

5.00

6.00

7.00

%

0

100

5.35

_6.104

5.735

4.00

Fragmentation spectrum of the semi-reduced form

(arrival time : 6.104 ms)

 

m/z

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1100

1150

%

0

100

515.62

136.05

265.12

279.12

461.25

299.15

_363.18

557.30

529.29

691.41

609.85

G R

C

C

H P A

C

G K Y Y S

C

SH

SH

S

S

[MH-Y]

2+

b

₅  

[MH

3

]

3+

iY

y

9  

y

8  

y

7  

y

112+  

b

62+  

a

72+  

a

₅2+  

b

52+  

YY-28

y

122+  

[MH-K-Y-Y]

b

72+  

[MH-Y-Y-S]

2+

a

5  

[MH-Y-Y]

2+

820.50

891.56

988.65

b

2  

b

4  

[MH

3

-H

2

O]

3+

[MH

3

-28]

3+

Time

(ms)

5.00

6.00

7.00

%

0

100

5.35

_6.104

5.735

4.00

19

See

poster #15

(J. Echterbille

et al

.)

for a new potential

application to

prevent the

reoxidation

•  Ion Mobility is a valuable tool for small molecules

analyses because of:

–  Spectra cleaning of trace residues (addition of a new orthogonal

separation of ion, removing the matrix background)

–  Provide a new potential identification criterion for screening and

identification for analytical purposes: the drift time

–  Get deeper insight into structural information compare to “regular” MS

•  Instrument can offer additional potentialities that are

under investigations to propose new strategies with

IM-MS

Thank you for your attention

Acknowledgments:

I would like to thanks:

–  The respective authors for providing figures (pesticide, venomics)

–  Romain Touilloux for his work during the pesticide screening project by IMS

–  Members of the LSM lab for valuable discussions

–  ANALIS SA/NV for my current financial support