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The usefulness of Ion Mobility-Mass Spectrometry for Small Molecules
Analysis
J. Fara, S. Goscinnyb, L. Jolya,b, R. Touillouxa, J. Echterbillea, L. Quintona , G. Eppea and E. De Pauwa
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
Plan
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: www.bowers.chem.ucsb.edu/theory_analysis/ion-mobility/index.shtml
• 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)
Different design of IMS
4
• 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 ≈ mgas
• Ω: collision cross-section (A²)
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 (NH3?)
(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
6 J. Far, G. Mazzucchelli, E. De Pauw , G. Eppe
Structural assignation of an isobar compound in a L and D,L- SeMet standard by ESI IM MS and MSE
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
• [C10H18N2O7Se + 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)]
OHNH
SeH+
O
OH
OH
O
NH3+
OH
O
Se
NH3+
OH
O
m/z: 181.9720 m/z: 176.0559
NH2+
O
OH
OH
O
OH
m/z: 88.0399 m/z: 269.9881
OHNH
O
O
OH
OHSe
NH3+
OH
O
OHNH
O OH
OH
Se
NH3+
OH
O
Om/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 (MS2), MS3 and MS4 data
8
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
35
1.07
72
356.
0181
360.
1632
36
0.96
86
363.
0983
359.0439
357.
0418
355.
0453
35
4.09
03
362.
0659
9
S.A.X
2nd LC dimension: Anion Exchange HPLC (PRPX-100)
25
10 t
o 25
0mM
CH
3CO
2NH
4 pH
5.5
50
75
0
1
2
3
4
10 20 30 40 50 60 70
sign
al (c
ps) 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
sign
al (x
10+
5 cps
)
time (minutes)
First LC dimension: Size Exclusion Chromatography
82Se 78Se
ICP Q MS
Preparative S.E.C
Se-rich yeast water extract
100 200 300 400 500 600 700 800 900 1000 m/z 0
350
700 741
Inte
nsity
, cou
nts
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 388.2612 449.6294
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:
10 J. Far, E. De Pauw Edwin, R. Lobinski, G. Eppe
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
100 266 135.9715
133.9702
84.0826 129.1065
164.9348 181.9741
359.0430 260.2022 222.0497
357.0492 280.0170
MS/MS 359 bin 138
269.9879 Noisy
m/z
%
0
100 152 176.0601
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
100 711 181.9741
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
11
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
12
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 Dri4 Tim
e (m
s)
m/z
Carbamate
Nitrogen pesNcide
Organophosphate
Pyrethroid
S. Goscinny, L. Joly, E. De Pauw, V. Hanot and G. Eppe
13
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
MS IMS
14
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 Poster 15 16
Drift time (ms)
Inte
nsity
(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 Poster 15
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 13
6.05
299.
15
186.
11 50
6.28
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+
b2
y122+ (b6-‐1)2+
(b7-‐1)2+ [MH-K-Y-Y]2+
y112+
[MH3]3+
(y9-‐1)2+
[MH3-H2O]3+
[MH3-H2O]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 51
5.62
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
b10 b9
b122+ b112+
b133+
b82+
b2
b102+
b92+ b8
b132+
[MH-P]2+ (y11-‐1)2+
y9-‐1
y1 y2
y3 y6
y7-‐1 y8-‐1
b5-‐1
(b6-‐1)2+
(b7-‐1)2+
[MH3]3+
iY
YY-28
YS-28
[MH3-H2O]3+
[MH3-28]3+
b5-‐1
Time (ms)
5.00 6.00 7.00
%
0
100 5.35 6.104
5.735
4.00
18
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 S SH S
[MH-Y]2+
b5
[MH3]3+
iY
y9 y8 y7 y112+
b62+
a72+ a52+
b52+
YY-28
y122+
[MH-K-Y-Y]
b72+
[MH-Y-Y-S]2+
a5 [MH-Y-Y]2+
820.
50
891.
56
988.
65
b2 b4
[MH3-H2O]3+
[MH3-28]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
Conclusions and perspectives
20
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
– The organizers to give us the opportunity to present these works
