Supplemental Material
Supporting Information
A Uniform Field Ion Mobility Study of Melittin and
Implications of Low-Field Mobility for Resolving Fine CrossSectional Detail in Peptide and Protein Experiments
Jody C. May and John A. McLean
Department of Chemistry; Center for Innovative Technology, Vanderbilt Institute for Integrative
Biosystems Research and Education; Vanderbilt Institute of Chemical Biology; Vanderbilt
University, Nashville, TN
Email: john.a.mclean@vanderbilt.edu
Supplemental Material
Details of the Helium Collision Cross Section Measurements
The procedure for calculating the collision cross section (CCS) from experimental uniform
field measurements involves three primary steps: (1) extraction of raw drift times from the ion
mobility arrival time distributions (ATD), (2) correcting the raw drift times for the non-mobility
time components, and (3) conversion of corrected drift times into the ion-neutral CCS using the
Mason-Schamp relationship [1], which is used in the following form:
3∙𝑍∙𝑒𝑐
2𝜋
𝐶𝐶𝑆 = ( 16∙𝑁 ) ∙ (𝑘
𝐵 ∙𝑇
1
2
1
𝑚𝑖𝑜𝑛 +𝑚𝑔𝑎𝑠 2
𝑉∙𝑡𝑑
𝑖𝑜𝑛 ∙𝑚𝑔𝑎𝑠
𝐿2
) ∙(𝑚
) ∙(
∙
273.15
𝑇
𝑃
∙ 760)
(Equation 1)
Here, Z is the integer charge state of the ion (unitless), ec is the constant for elementary charge
(1.60217657 x 10-19 C), N is the gas number density (molecules/m3), kB is the Boltzmann constant
(1.3806488 x 10-23 J•K-1), mion is the ion mass (Da), and mgas is the helium gas mass (4.0026029
Da).
For step 1 of the CCS workflow, an ion mobility spectrum is extracted from a narrow m/z region
and specific drift time ranges containing peak features of interest are then selected and processed
for centroid time values. In the case where the peak resolution is insufficient for the software to
calculate a centroid value, the peak centroid is determined by visual inspection, which allows the
CCS of low abundance peak features to be estimated at a cost of reduced measurement precision.
While somewhat subjective, we find that manual determination of peak centroids still provides
good CCS precision (well below 2%). In step 2, these raw drift times are compiled for a range of
drift fields (Table S1) in order to determine the non-mobility time component of the experiment.
Because the non-mobility time component arises from the ion’s transit through several pressure
and voltage regimes throughout the instrument (and thus has both ion mobility and mass time-offlight contributions), this value cannot be accurately determined a priori and so the stepped drift
field method is used. It was found that the higher fields resulted in an abrupt loss of resolution for
closely spaced peak features, and so only the seven lowest field values (1.9, 3.2, 4.5, 5.8, 7.1, 8.3,
and 9.6 V/cm) were subsequently used for purposes of the drift time correction and CCS
calculation. This observation is discussed in the manuscript. For each peak feature, the raw drift
times (y-axis) are plotted against the inverse drift voltages (1/V, x-axis) and the time intercept is
extrapolated from a linear fit to the data. In all cases, the coefficient of determination (R2) of the
linear fit of drift time vs. 1/V was better than 0.999, suggesting that the experimental precision is
high and that the ion system is governed by low field conditions where the drift field does not
affect the ion mobility. The time intercept extracted from this procedure represents the time ions
spend outside of the ion mobility drift region and is subtracted from the raw drift time in order to
determine the actual ion mobility drift time of each peak feature. The rationale for this analysis is
that the ion drift time will approach zero in the limit of infinite electric field (1/V = 0), leaving
only the non-mobility time component. For the final step (step 3), once the actual drift times are
determined, the CCS can be calculated directly from the Mason-Schamp relationship (equation
1).
Supplemental Material
Drift Fields Investigated and their Corresponding Voltage and E/N Values
Drift Field (V/cm):
1.9
3.2
4.5
5.8
7.1
8.3
9.6
10.9
12.2
Drift Voltage (V):
151
251
351
451
551
651
752
852
952
E/N (Td):
1.5
2.6
3.6
4.6
5.6
6.6
7.6
8.7
9.7
E/N values correspond to 4.000 ± 0.001 Torr and 306.5 ± 0.5 K.
Table S1. A summary of the drift fields investigated in this work. The corresponding drift
voltages and E/N values (expressed as Townsend units, Td) are also provided.
In uniform field ion mobility, the characteristic ion mobility energy parameter, E/N, can be
directly calculated using the following equation:
𝐸
𝑁
𝑉
= 𝐿∙𝑁 ∙ 1017
(Equation 2)
Here V is the drift voltage (V), L is the length of the drift region (78.1 cm), and N is the gas number
density (molecules/cm3), determined from the ideal gas law. The 1017 multiplier in Equation 2
converts V·cm2 into Td units (10-17 V·cm2) [2]. For this study, resolution of finer detail within
peptide peak features is observed below an E/N value of ca. 4 Td, although successfully operating
at these corresponding low field values is contingent on ability to obtain good ion statistics, which
in turn depends on the abundance of the ion as well as the sensitivity of the instrumentation utilized.
Supplemental Material
Helium Collision Cross Section (Å2)
Melittin Ion
Form
0% MeOH
100% H2O
25% MeOH
75% H2O
50% MeOH
50% H2O
75% MeOH
25% H2O
100% MeOH
0% H2O
[M+H]1+
1A
m/z 2845.76
469 ± 30 (3) a
2A
[M+2H]2+
2B
m/z 1423.38 2C
2D
355.0 ± 1.1 (7)
385.1 ± 3.6 (7)
449.9 ± 2.9 (7)
--
355.0 ± 2.6 (7)
390.3 ± 2.9 (7)
449.6 ± 3.8 (7)
--
356.1 ± 1.0 (7)
393.8 ± 1.7 (7)
450.8 ± 3.0 (7)
--
359.1 ± 1.8 (7)
403.7 ± 1.5 (7)
451.5 ± 4.9 (7)
--
-419.7 ± 8.9 (6)
-469.7 ± 13.5 (7)
[M+3H]3+
m/z 949.25
3A
3B
3C
405.9 ± 1.0 (7)
488.4 ± 1.5 (7)
523.0 ± 1.7 (7)
407.2 ± 0.7 (7)
487.8 ± 1.1 (7)
523.3 ± 1.6 (7)
404.7 ± 1.0 (7)
486.6 ± 0.9 (7)
523.6 ± 0.4 (7)
405.4 ± 0.7 (7)
487.6 ± 1.6 (7)
525.8 ± 1.1 (7)
407.8 ± 4.8 (7)
489.0 ± 7.4 (7)
526.6 ± 7.7 (7)
[M+4H]4+
m/z 712.20
4A
4B
4C
541.9 ± 0.8 (7)
593.5 ± 2.7 (4)
--
543.2 ± 0.4 (7)
595.3 ± 1.2 (5)
--
540.8 ± 0.7 (7)
595.1 ± 1.3 (6)
--
540.6 ± 0.7 (7)
611.0 ± 3.2 (7)
657.5 ± 4.0 (7)
541.7 ± 7.1 (7)
602.9 ± 16.4 (7)
--
[M+5H]5+
m/z 569.96
5A
5B
5C
5D
605.3 ± 5.6 (7)
631.7 ± 1.8 (7)
---
595.9 ± 4.9 (7)
628.4 ± 1.0 (7)
---
601.1 ± 2.1 (6) 602.0 ± 8.7 (7)
623.2 ± 0.9 (6) 626.3 ± 1.0 (7)
645.3 ± 12.9 (6)
-670.0 ± 5.6 (7)
--
605.7 ± 6.8 (7)
629.9 ± 9.6 (7)
646.0 ± 7.1 (7)
669.0 ± 11.0 (7)
--
485 ± 10 (5) b
--
--
a Ions
generated by MALDI. The CCS was measured on a periodic DC-field drift tube instrument [3].
Ions generated by MALDI. The CCS was measured on a home-built drift tube instrument in the authors’
laboratory, which has been described previously [4].
b
Table S2. A summary of CCS measurements for all signifcant peak features observed
for various charge states of melittin. Singly-protonated melittin CCS measurements were
obtained using MALDI, and categorized based on solvent preparation conditions. Values
highlighted in bold represent the most abundant peak feature for the corresponding
solvent and charge state. All CCS values exhibit an RSD of better than 2%.
Supplemental Material
Precision and Accuracy of Helium Collision Cross Section Measurements
The IM-MS instrumentation used in this study was originally developed to support
nitrogen-based CCS measurements. In order to determine the precision and accuracy for obtaining
helium CCS values, a series of tetraaklylammonium (TAA) salts were analyzed. The helium CCS
values of these salts have previously been measured using a confining-RF drift tube instrument
[5]. Helium CCS results for the TAA salts are summarized in Table S3. The percent relative
standard deviation (% RSD) for all CCS values is better than 1%, with a corresponding agreement
to literature to be better than 0.5% based on the relative difference to the mean. These results are
consistent with the performance of the instrument for nitrogen studies [6], and suggest good
precision and accuracy can also be expected for the helium CCS measurements.
Helium Collision Cross Sections (Å2) for Tetraaklylammonium Salts
Ammonium
Salt
Mass
(Da)
CCS (Å2),
This work a
%
RSD
CCS (Å2),
Literature b
% Difference,
Relative c
TAA2
130.16
64.9 ± 0.2 (6)
0.3%
65.9 (0.0%)
0.4%
TAA3
186.22
89.0 ± 0.4 (6)
0.4%
88.9 (0.1%)
0.0%
TAA4
242.28
111.8 ± 0.6 (6)
0.5%
111.2 (0.0%)
0.1%
TAA5
298.35
134.1 ± 0.8 (6)
0.6%
133.5 (0.0%)
0.1%
TAA6
354.41
155.6 ± 0.9 (6)
0.6%
154.9 (0.2%)
0.1%
TAA7
410.47
175.2 ± 1.1 (6)
0.6%
174.5 (0.5%)
0.1%
TAA8
466.54
192.6 ± 1.6 (6)
0.8%
194.3 (0.0%)
0.2%
TAA10
578.66
223.8 ± 1.8 (6)
0.8%
--
--
a.
Values in parenthesis represent the number of measurements.
Values obtained from reference [5]. Values in parenthesis are the % RSD.
c.
The difference relative to the mean of the two values, expressed as a percentage.
b.
Table S3. A summary of CCS measurements obtained for the TAA salts. A
corresponding comparison of the relative percent difference of measured values to ones
reported in the literature are also provided.
Supplemental Material
Figure S1. An IM-MS spectrum of melittin electrosprayed from pure aqueous solvent.
The HP-621, HP-921, HP-1221, and HP-1521 ions which appear in the spectrum are
mass calibrants (Agilent ESI tune mixture). The mass spectrum does not exhibit any
signficant ion signal between 1700 and 3200 (range not shown).
Supplemental Material
Details of the Experiments for Evaluating the Presence of Isobaric Aggregates
The existence of aggregates (multimers) which are isobaric with the mass-to-charge can
confound the interpretation of peptide and protein ion mobility data [7]. Since multimer formation
is directly linked to solution concentration, a series of experiments were conducted at three
different melittin peptide concentrations (1, 10, and 100 μM). These comparisons were conducted
in pure water, as aqueous conditions have explicitly been shown to promote the formation of
melittin aggregates [8]. Narrow m/z windows were isolated for each observed melittin charge
state ([M + nH]n+, where n = 2 to 5) and these IM-MS spectra were examined for evidence of
multimers. Based on an analysis of the mass spectral data, there is no evidence that multiplycharged multimers exist isobaric to the melittin charge states, as the isotope spacing for each ion
species correspond to their presumed charge state (Figures S2-S4). The rationale here is that
isobaric multimers would exist at higher charge state values. For example, m/z 1423.38 could arise
from the [M + 2H]2+ ion, but also from the [2M + 4H]4+ or the [3M + 6H]6+ ions. Since mass
spectrometry measures the mass-to-charge ratio of the ion, the presence of these higher charge
state ions would be clearly indicated in the isotope spacing. This does not preclude the possibility
that such multimers exist and dissociate prior to MS analysis, as multimers would be
noncovalently-linked and thus subject to a weak dissociation energy threshold. An additional
analysis was conducted in which the ion mobility ATDs from each peptide concentration was
overlaid and examined for evidence of concentration-specific changes in individual peak features.
The relative abundance and shape of peak features for all charge states remains largely unaffected
by changes in solvent concentration across the two orders-of-magnitude investigated (Figure S5),
suggesting that there is no significant contribution to multimers up to 100 μM peptide
concentration. A slight decrease in the peak height of the “2A” peak feature is observed at higher
concentration, although this is interpreted as not significant as the total abundance of the [M +
2H]2+ ion mobility ATD is low (ca. two orders-of-magnitude lower than the [M + 3H]3+ ATD
based on peak height). Thus, there is no direct evidence of isobaric multimers in this study. To be
certain, the peptide concentration was limited in this study to 10 μM, which represents the lowest
concentration from which sufficient ion mobility spectra could be obtained for all charge states
investigated.
Supplemental Material
Figure S2. IM-MS spectra obtained from 1 μM sample concentration of melittin peptide
in pure aqueous solvent. Spectra represent IM-MS windows for the (A) [M + 2H]2+, (B)
[M + 3H]3+, (C) [M + 4H]4+, and (D) [M + 5H]5+ ions. All spectra are obtained from a single
experimental run with ion mobility separations at 3.6 Td (4.5 V/cm in 4.000 Torr helium
drift gas). At this low concentration, traces of a singly-charged interferent is observed in
the [M + 4H]4+ spectra (C), and this is considered when extracting the ATD and obtaining
drift time measurements for the CCS. Ions in panels (A) and (D) are below the limits of
detection at this sample concentration.
Supplemental Material
Figure S3. IM-MS spectra obtained from a 10 μM sample concentration of melittin peptide
electrosprayed from pure aqueous solvent. IM-MS windows represent the (A) [M + 2H]2+,
(B) [M + 3H]3+, (C) [M + 4H]4+, and (D) [M + 5H]5+ ions. All spectra are obtained from a
single experimental run with ion mobility separations conducted at 3.6 Td (4.5 V/cm in
4.000 Torr helium drift gas). At this concentration, there is sufficient abundance of all four
ion charge states for ATD feature comparisons and CCS measurements.
Supplemental Material
Figure S4. IM-MS spectra obtained from a 100 μM sample concentration of melittin
peptide in pure aqueous solvent. Spectra represent IM-MS windows for the (A) [M +
2H]2+, (B) [M + 3H]3+, (C) [M + 4H]4+, and (D) [M + 5H]5+ ions. All spectra are obtained
from a single experimental run with ion mobility separations at 3.6 Td (4.5 V/cm in 4.000
Torr helium drift gas). The isotope spacing observed in each mass spectra are indicative
of the assigned charge state, indicating no contribution of ions originating from highercharged oligomers.
Supplemental Material
Figure S5. An overlay of ion mobility spectra obtained at 4.6 Td (5.8 V/cm) for different
sample concentrations in pure aqueous solvent. Each panel represents a different charge
state of melittin. All spectra are converted to CCS and normalized to the same maximum
abundance to facilitate comparison. Significant peak features observed at this solution
composition are noted. For all charge states, there is no significant change in the ATDs
across two orders of magnitude in sample concentration, suggesting no strong
contribution from multimers at these concentrations. The lower apparent abundance of
the 2A feature at higher concentration is not reproducible at the other drift fields, which is
attributed to the unreliability of peak height for comparing abundances of poorly-resolved
features.
Supplemental Material
Figure S6. A comparison of the number of samples (bins) across the [M + 2H]2+ ion
mobility ATD for (A) 9.7 Td (12.2 V/cm), and (B) 3.6 Td (4.5 V/cm). Measurements at
higher drift fields results in compression of the mobility ATD to a narrow time range, which
subsequently results in less sampling bins across the ATD. The panels at right expand
the region of a mostly-resolved peak feature (2C), indicating ca. 16 bins across this peak
at 9.7 Td. This is approximately half of the bins at 3.6 Td, however, this sampling
resolution is still sufficient to discern details within the ATD.
While not explicitly explored in this study, the issue of insufficient sampling at higher fields
can be experimentally evaluated. Using the present commercial IM-MS, the total bins across each
ion mobility spectrum is adjustable by varying the ion mobility acquisition rate (denoted as “IM
Transient Rate” in the Masshunter acquisition software). By selecting different IM acquisition
rates, the ATDs can be directly compared for evidence of insufficient sampling.
Supplemental Material
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