Supplemental: A Mechanism for Ionization of Nonvolatile Compounds in
Mass Spectrometry: Considerations from MALDI and Inlet Ionization
Sarah Trimpin,1* Beixi Wang,1 Ellen D. Inutan,1 Jing Li,1 Christopher B. Lietz,1
Andrew Harron, 2 Vincent S. Pagnotti,2 Diana Sardelis,2 Charles N. McEwen2*
1
Wayne State University, Department of Chemistry, Detroit, MI
2
University of the Science, Department of Chemistry and Biochemistry, Philadelphia, PA
Caveats of the proposed model
All observation made for inlet ionization are not easily explained by a charged
cluster/droplet model. For example, at low inlet temperature in AP-LSI and MAII ion
formation can be observed for many seconds after the sample enters the inlet. The
results displayed in Figure 3 demonstrate not only that analyte ion abundance increases
with increasing temperature as described previously,1 but that the time over which ions
of the analyte are observed is reduced significantly at higher temperature. A surface
must be involved in the long ionization time. For volatile compounds, vaporization and
chemical ionization by ions formed in the inlet explains the result, but for nonvolatile
compound it is not readily obvious how clusters are formed by a slow process involving a
surface. We speculate that matrix/analyte accumulates on a cool surface and particles
are slowly released by bubble formation or surface splintering. Similarly, it is not obvious
how a cluster mechanism can be involved in inlet ionization of pure myoglobin powder
(Figure 2B). One possibility that might involve charged droplets is residual water in the
myoglobin particle acts to produce ions in a SAII-like process. Alternatively, the
myoglobin particles could become charged by surface interactions (e.g. static charge)
and the combination of the repulsive electrostatic force and thermal energy is sufficient
to release myoglobin ions from the surface. At this time there are no data to support
these arguments.
Another problematic example is the observation that in LP laser ablation, using
the LSI matrix 2-NPG, multiply charged ions are only observed for proteins but not
peptides (Figure S10). Switching between IP-LSI and IP-MALDI (Figure 1) is yet only
possible using the matrix 2,5-DHAP and only for peptides but not proteins (Figure S9);
the problem being the difficulty of producing dominantly singly charged ions of the higher
molecular weight polypeptides under IP conditions.
The higher tendency for larger
polypeptides to produce multiply charged ions is also observed in AP-LSI and MAII
where the MALDI matrix sinapinic acid, with the inlet temperature >425º C produces
dominantly singly charged ions of the peptide angiotensin I but multiply charged ions in
low abundance with small proteins (Figure S3,I). Interestingly, lysozyme does not
produce multiply charged ions with sinapinic acid but when 5% 2-NPG is added,
abundant highly charged ions are observed (Figure S3, IID and IIE). One can speculate
that the 2-NPG crystallizes separate from the sinapinic acid and carries along some of
the lysozyme.
Additionally, in AP-LSI and MAII of peptides and proteins, it appears that the
higher the molecular weight, the higher the inlet temperature required for observation of
multiply charged analyte ions as well as for maximum analyte ion abundance (Figure
S12).
In a cluster model, the higher temperature requirement in LSII could represent
the change from ion evaporation2 to the residue mechanism3 as the molecular weight of
analyte increases, or it may be that larger ablated analyte molecules result in laser
ablation ejection of larger matrix/analyte droplets as previously reported4 requiring
increased energy for desolvation. Another possibility is that higher thermal energy is
necessary for ion evaporation of larger compounds.
While these observations still need explanation, at this point, they do not suggest
an alternative mechanism for ion formation.
Scheme 1. Representation of the different inlet ionization methods: A) AP-LSI, B) MAII,
and C) SAII at atmospheric pressure, and D) LSI-TOF in reflection geometry.
A. LSII
~ 1 - 3 mm
Glass slide
Laser
beam
+ + ++
+
++
+++ +++++ +
+++ +
+
+ +++
++
+
Matrix/analyte clusters
Flow entrapment
Matrix/
analyte Mass spectrometer
crystals orifice
B. MAII
To
vacuum
Glass slide
++
++++ ++++ +++
++ +++++
+ +
+
++ ++
+
Heated transfer
capillary
Matrix/
analyte Pressure drop
crystals
C. SAII
To
vacuum
To
vacuum
+
+ + +
+ +
+ +
+ +
+
Heated
transfer
capillary
Pressure drop
D. LSIV
Laser
beam
+
+
Glass Slide
+ +
+ +
+
+ ++
+
+
+ + ++ +
++ +
Matrix/analyte
Matrix/analyte
clusters
crystals
Heated
transfer
capillary
Pressure drop
517.26
100
K K F L L P E P T P L S P S R R
+447
90
1036.61
80
Relative Abundance
70
60
50
40
30
c2
z2
315.23
z5
Z152+
Z132+
z14
c1
825.34
c14 c15 z15
c10
550.37
1521.87
20 146.19
1927.62
c11
1755.96
z1
c3
690.45 963.48
159.07 421.32
1230.95 1408.81 z1652.16
10
13
c
Z82+
13
1372.66
0
200
400
600
800
1000
m/z
1200
1400
1600
1800
2000
Figure S1. AP-LSI-ETD-MS/MS mass spectrum of O-GlcNAc peptide standard using
2,5-DHAP as matrix acquired on a LTQ, mass spectrometer modified to accommodate a
Finnigan 4500 CI source (Thermo Electron) placed at the rear of the mass spectrometer
instrument. The inlet transfer capillary was heated at 350 C.
I. CHCA
II. SA
A) 450 °C
A) 450 °C
+1
1297.69
1:1 analyte:matrix layered spot
+2
648.85
80
60
40
20
0
60
1045.77
40
20
648.93
+2
80
+3
60 433.01
+1
40
1296.69
20
0
80
60
464.26
649.43
875.68
20
1079.69
B) 400 °C
1:2 analyte:matrix layered spot
1:2 analyte:matrix layered spot
761.43
80
611.43
929.60
60 433.26
649.43
1228.78
547.26
20
500
700
1327.86
100 (2)
900
m/z
1100
1300
Relative Abundance
100 (2)
Relative Abundance
+2
40
0
B) 400 °C
0
+1
1296.78
1:2 analyte:matrix layered spot
100 (2)
Relative Abundance
Relative Abundance
80
0
1:2 analyte:matrix layered spot
100 (2)
40
1296.61
100 (1)
Relative Abundance
Relative Abundance
100 (1)
+1
1:1 analyte:matrix layered spot
80
789.60
60 469.26
648.93
1051.61
1273.94
893.60
40
20
0
500
700
900
m/z
1100
1300
Figure S2. MAII-MS mass spectra of 2 pmol µL-1 angiotensin I prepared in (1) 1 µL
analyte solution and 1 µL matrix solution and (2) 1 µL analyte solution and 2 µL matrix
solution and 1 µL was air dried on a spatula and tapped against the inlet with I) CHCA
and II) SA acquired at A) 450 °C and B) 400 °C inlet capillary temperature using an
LTQ-Velos mass spectrometer.
60
40
+1
20
1296.69
500
700
B) Bovine Insulin
100
900
1100
6.26 e2
+3
1911.73
20 660.82
1000
1400
1800
2200
2600
C) Ubiquitin +8
Relative Abundance
60
40
+11
779.55
+5
1713.27
2072.00
20
0
600
1000
D) Lysozyme
1400
1800
2560.82
2200
2600
+11
1310.00
100
500
700
2.03 e2
+4
1434.55
40
20
+5
1147.45
600
1000
2442.64
859.82
2870.18
20
1800
m/z
III. 95% CHCA: 5% 2-NPG
E) Lysozyme
100
2200
2600
Relative Abundance
60
40
20
288.50
0
200
+15
954.93
+16
895.35
+17
842.35
+18
796.35
+19
600
1000
m/z
3000
+7
1224.55
60
+5
1713.82
+8
1071.82
40
+9
952.55
+10
857.55
20
600
2142.36
1000
1400
1800
1854.64
2644.27
2200
2600
3000
1.09 e2
2355.82
60
1203.73
909.82
40
20 614.64
0
3000
2788.82
80
600
1000
1400
1800
2200
2600
3000
IV. 95% SA: 5% 2-NPG
E) Lysozyme
+12
1192.94
8.95E3
+13
1101.52
+14
1022.77
80
Relative Abundance
1118.55
40
1400
2600
7.62 e2
D) Lysozyme
+11
1301.27
+10
1431.36
+9
1590.11 +8
1789.12
1400
1800
100
Relative Abundance
Relative Abundance
60
1000
2200
1667.36
1852.55 2150.45
600
1800
+6
1428.18
80
0
2868.18
1400
100
80
0
1300
1.38 e3
60
C) Ubiquitin
3000
1100
+3
1911.82
1669.73
550.45
900
80
100
+7
1224.45 +6
1428.55
+10
857.45
80
20
0
3000
3.27 e2
1071.55
+9
952.55
100
531.36
40
100
Relative Abundance
+5
1147.82
600
60
B) Bovine Insulin
40
0
+2
649.18
80
0
1300
+4
1434.36
80
60
1.06 e2 +1
1296.64
100
80
0
II. SA
A) Ang. I
Relative Abundance
Relative Abundance
+3
100 433.34
Relative Abundance
8.18 e2
+2
648.93
Relative Abundance
I. CHCA
A) Ang. I
80
2.27E3
+12
1192.86
+11
1301.27
225.17
60
+13
1101.35
+10
1431.44
40
368.59
20
0
200
+9
1590.45
+8
1789.12
+14
760.68 1023.02
663.51
600
1000
m/z
1400
1800
Figure S3. MAII-MS mass spectra of 5 pmol µL-1 of A) angiotensin I, B) bovine insulin,
C) ubiquitin, and D) lysozyme with I) CHCA and II) SA acquired at 450 °C inlet capillary
temperature using an LTQ-Velos mass spectrometer. E) Mass spectra of lyzozyme
using binary matrix mixture of III) 95% CHCA: 5% 2-NPG and IV) 95% SA: 5% 2-NPG.
Analyte/matrix spot was prepared in 1:2 volume ratio using the layer method on a glass
plate and air dried.
+9
A) 120 °C
952.70
100
2.66e4
+10
857.43
+8
1071.65
%
+11
779.58
663.52
+7
1224.44
+6
+5
1428.33
+12
1713.75
607.45
0
B) 40 °C
857.34
100
1071.37
164
760.60
%
951.01
1224.45
1430.03 1614.16
714.82
1705.93
663.51
0
600
800
1000
1200
m/z
1400
1600
1800
Figure S4. AP-LSI-MS mass spectra of ubiquitin prepared using the layer method with
2,5-DHAP in 1 µL analyte solution and 4 µL matrix solution at A) 120 C and B) 40 C
source temperature on a SYNAPT G2 mass spectrometer.
I. Visible 532 nm
A. Delipified Tissue
1)
3) Extracted Drift Times
2)
BW_2011_05_05 STAINLESS STEEL 04.raw : 1
+2
m/z
917.62
1000
2
4
6
Drift time (ms)
8
BW_2011_05_05 STAINLESS STEEL 04.raw : 1
+3
612.10
m/z 917
50%
+2
+3
m/z 612
%
0
100
50%
3
4
5
6
Drift time(ms)
2
7
4
6
Drift time (ms)
8
B. Synthesized Neuropeptide
1)
3) Extracted Drift Times
2)
BW_2011_05_04 STAINLESS STEEL 03.raw : 1
m/z 917
+2
1000
m/z
917.61
50%
+2
2
4
6
Drift time (ms)
8
BW_2011_05_04 STAINLESS STEEL 03.raw : 1
612.08
+3
+3
m/z 612
%
0
100
50%
3
4
5
6
Drift time(ms)
2
7
4
6
Drift time (ms)
8
II. IR 1064 nm
A. Delipified Tissue
BW_2011_05_05 STAINLESS STEEL 16.raw : 1
1)
3) Extracted Drift Times
2)
BW_2011_05_05 STAINLESS STEEL 16.raw : 1
917.68
+2
+2
m/z
1000
2
4
6
Drift time (ms)
8
BW_2011_05_05 STAINLESS STEEL 16.raw : 1
+3
612.47
m/z 917
50%
+3
m/z 612
50%
0
%
100
3
4
5
6
Drift time(ms)
2
7
4
6
Drift time (ms)
8
B. Synthesized Neuropeptide
BW_2011_05_05 STAINLESS STEEL 17.raw : 1
1)
3) Extracted Drift Times
2)
BW_2011_05_05 STAINLESS STEEL 17.raw : 1
m/z 917
918.1859
+2
+2
1000
50%
+3
4
6
Drift time (ms)
8
BW_2011_05_05 STAINLESS STEEL 17.raw : 1
+3
m/z 612
0
50%
%
100
612.1412
m/z
2
3
4
5
6
Drift time(ms)
7
2
4
6
Drift time (ms)
8
Figure S5. AP-LSI-IMS-MS of the neuropeptide, MBP, from A) delipified mouse brain
tissue and B) synthesized MBP peptide using 2,5-DHAP matrix at (I) 532 nm and (II)
1064 nm wavelengths. Mass spectra (left panel), 2-D plots of drift time vs. m/z (middle
panel) and extracted drift times for +2 and +3 ions (right panel) are displayed.
A) Leucine Enkephalin
1) UV 337 nm
329.03
100
2.04e3
[M+H]+
556.24
%
0
2) Visible 532 nm
556.33
100
%
9.96e3
594.28
0
3) IR 1064 nm
556.23
1.09e4
100
%
0
329.02
100
300
500
m/z
B) Bovine Insulin
1) UV 337 nm
100
900
+4
1434.18
+5
1147.55
%
700
1.07e3
+3
1912.19
+6
962.46
663.47
0
2) Visible 532 nm
1434.59
100
%
1.49e3
1147.67
1912.75
956.72
0
3) IR 1064 nm
2.10e3
1434.37
100
1147.69
%
1911.44
962.54
0
700
900
1100
1300
m/z
1500
1700
1900
C) Ubiquitin
1) UV 337 nm
100
+9
952.64
+10
+8
857.28
1071.47
+11
6.34e3
+7
1224.2574 +6
1428.31
779.43
%
+5
1714.33
0
2) Visible 532 nm
100
%
952.65
1071.59
857.47
1224.55
779.71
1428.79
726.60
4.32e3
1713.73
0
3) IR 1064 nm
100
2.42e3
952.60
1071.54
857.33
%
1224.48
779.66
1428.24
0
500
700
900
1100
1300
m/z
1500
1714.27
1700
1900
Figure S6. AP-LSI mass spectra of A) leucine enkephalin, B) bovine insulin, and C)
ubiquitin using 2,5-DHAP as matrix acquired on the SYNAPT G2 at source temperature
of 150 ºC using various wavelengths at (1) UV 337 nm, (2) Vis 532 nm, and (3) IR 1064
nm.
[M+H]+
703.62
%
100
[M+Na]+
725.60
[M+K]+
741.56
0
685
695
705
715
725
m/z
735
745
[M+Cu]+
765.53
755
765
775
Figure S7. Extracted mass spectrum of sphingomyelin from the 2-dimensional plot
acquired using 2,5-DHAP matrix on the SYNAPT G2 mass spectrometer. A fabricated
Cu tube was used as the inlet capillary attached to the skimmer with a source
temperature of 150 C. Copper adducts were observed using this inlet tube.
A
2.88e3
+6
955.92
%
100
+5
1146.89
+4
1433.37
+3
1911.46
+7
827.04
m/z
0
1434.08
689
B
%
100
1147.24
1911.43
956.43
0
700
900
1100
1300
1500
1700
1900
m/z
Figure S8. SAII-MS of 5 pmol µL-1 bovine insulin in 50:50 MeOH:water with 1% acetic
acid acquired on the SYNAPT G2 mass spectrometer using a homebuilt SAII device
where the inner cone is modified by inserting a metal tube through the cone inlet: A)
using an obstruction after the tube outlet and B) without obstruction. The source
temperature was set at 150 C and no additional heat applied to the inlet tube.
+5
1147.77
A
3.71e3
1
+4
1434.46
%
100
+6
956.48
0
B
1600
2000
2400
0
m/z
588
1500
2500
3500
4500
+2
%
2
5500
m/z
1.74e4
+3
+4
0
2800
+3
1912.31
+5
100
1200
1
%
100
800
+4
1434.51
+3
1911.93
1500
+1
2500
3500
4500
5500
m/z
Figure S9. IP-LSI mass spectra of 1 pmol µL-1 bovine insulin with 2-NPG matrix
prepared using droplet method in 1 µL analyte solution and 1 µL matrix solution and
acquired using A) ESI like settings and B) tuned in as MALDI settings on SYNAPT G2
mass spectrometer with (1) low laser fluence setting of ‘160’ and ‘175’ and (2) high laser
fluence setting of ‘250’.
A
1.18e4
%
100
+4
1434.63
+5
1147.91
+3
1912.10
+6
0
B
1912.03
100
1.74e4
%
1434.31
+2
2867.65
0
1000
2000
3000
m/z
4000
5000
6000
Figure S10. IP-LSI mass spectra of 1 pmol µL-1 bovine insulin with 2,5-DHAP as matrix
prepared using the dried droplet method applying 1 µL analyte solution and 1 µL matrix
solution and acquired using A) ESI settings and B) MALDI settings on the SYNAPT G2
mass spectrometer. Laser fluence was set to “200”.
1834.11
A
+1
800
Intens. [a.u.]
600
400
200
+2
918.06
0
1000
1200
1400
1600
1800
2465.27
B
+1
Intens. [a.u.]
100
80
60
40
+2
1234.62
20
0
1000
1400
1800
m/z
2200
2600
Figure S11. LSI-TOF mass spectra of A) N-acetylated myelin basic protein fragment (MBP)
and B) adrenocorticotropic hormone (ACTH) prepared using the dried droplet method with
2-NPG matrix in 1 µL analyte solution and 1 µL matrix solution and acquired in reflectron
mode on a MALDI-TOF-TOF mass spectrometer.
(1)
(2)
(3)
(2)
(1)
(3)
Figure S12. AP-LSI Inlet temperature vs. ion abundance graph for (1) bradykinin +2
charge state, (2) Insulin average abundance of charge states +3 to +5, (3) lysozyme
average abundance for charge states +8 to +10. The peak abundance for bradykinin
occurs at ca. 290 ºC, for insulin at 345 ºC, and for lysozyme at 395 ºC.
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Dole, M.; Mack, L. L.; Hines, R. L.; Mobley, R. C.; Ferguson, L. D.;
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