Shift reagents for multidimensional ion mobility spectrometry-mass spectrometry analysis of complex peptide mixtures: Evaluation of 18-crown-6 ether complexes

Article abstract of DOI:10.1021/ac200892r

18-Crown-6 ether (18C6) is evaluated as a shift reagent for multidimensional ion mobility spectrometry-mass spectrometry (IMS-IMS-MS) analyses of tryptic protein digests. In this approach, 18C6 is spiked into the solution-phase mixture and noncovalent peptide-crown ion complexes are formed by electrospraying the mixture into the gas phase. After an initial mobility separation in the first IMS drift region, complexes of similar mobility are selected and dissociated via collisional activation prior to entering the second drift region. These dissociation products (including smaller complexes, naked peptide ions, charge transfer products, and fragment ions) differ in mobility from their precursor ion complexes and (in favorable cases) from one another, allowing the mixture to resolve further in the second IMS region. We estimate an IMS-IMS peak capacity of ～2400 when shift reagents are employed. The approach is illustrated by examining a tryptic digest of cytochrome c and by identifying a peptide out of a complex mixture obtained by digestion of human plasma proteins. Disadvantages arising from increased complexity of data sets as well as other advantages of this approach are considered.

Full text of DOI:10.1021/ac200892r

ARTICLE
pubs.acs.org/ac
Shift Reagents for Multidimensional Ion Mobility Spectrometry-Mass
Spectrometry Analysis of Complex Peptide Mixtures: Evaluation of
18-Crown-6 Ether Complexes
Brian C. Bohrer and David E. Clemmer*
Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States
ABSTRACT:
18-Crown-6 ether (18C6) is evaluated as a shift reagent for multidimensional ion mobility spectrometry-mass spectrometry (IMS-
IMS-MS) analyses of tryptic protein digests. In this approach, 18C6 is spiked into the solution-phase mixture and noncovalent
peptideꢀcrown ion complexes are formed by electrospraying the mixture into the gas phase. After an initial mobility separation in
the first IMS drift region, complexes of similar mobility are selected and dissociated via collisional activation prior to entering the
second drift region. These dissociation products (including smaller complexes, naked peptide ions, charge transfer products, and
fragment ions) differ in mobility from their precursor ion complexes and (in favorable cases) from one another, allowing the mixture
to resolve further in the second IMS region. We estimate an IMS-IMS peak capacity of ∼2400 when shift reagents are employed.
The approach is illustrated by examining a tryptic digest of cytochrome c and by identifying a peptide out of a complex mixture
obtained by digestion of human plasma proteins. Disadvantages arising from increased complexity of data sets as well as other
advantages of this approach are considered.
on mobility spectrometry (IMS) coupled to mass spectro-
metry (MS) is becoming widely used in the analysis of complex
be induced, the multidimensional separation offers no advantage
relative to a single IMS separation.
I
mixtures, such as those encountered in the emerging areas of
proteomics, metabolomics, and glycomics.1ꢀ15 One factor that
limits this combination is that IMS separates ions based on
differences in their sizes (or average collision cross section) while
MS separates based on differences in ion masses, and these
properties are intrinsically correlated. In several recent reports,
we have described the development of multidimensional IMS-
MS (IMS-IMS-MS and IMS-IMS-IMS-MS)16,17 analyses of
peptides and proteins.17ꢀ21 With these techniques, we aim to
alter the intrinsic correlation of ion size and mass by changing the
ions’ shapes. This can improve the detection of low-intensity
signals and increase the analytical peak capacity (important for
many-component mixtures).22 Merenbloom et al. demonstrated
a peak capacity for an IMS-IMS separation of peptides (prior to
MS) in excess of 1000.22 While this is substantial, the approach is
fundamentally restricted by the extent to which structures change
upon collisional activation. In favorable cases, cross sections can
be altered by as much as (11% upon activation.22 However, for
many ions, collisional activation results in little change in the
overall ion shape (often only a few percent change in cross
section is observed).22 In cases where no change in mobility can
In this paper we use an IMS shift reagent23,24 to increase the
differences in mobility between analyte ions studied before and
after activation. Shift reagents are molecules that form noncova-
lent adducts with analyte species (either in solution or during the
ionization process) to produce ion complexes.23,24 We have
previously examined the use of 18-crown-6 ether (18C6) as a
shift reagent and illustrated a method that utilizes a single IMS
dimension to resolve three isobaric tripeptides that form peptideꢀ
crown complexes.23 In an IMS-IMS analysis using shift reagents,
analytes initially separate as a mixture of adduct ions and are
subsequently collisionally activated such that smaller complexes
(or naked ions) can be separated in a second IMS region.
Below, we investigate the advantages of shift reagents with
peptides generated by digestion of proteins with trypsin.25
Proteolytic digestion is a common method of generating peptide
mixtures for “bottom-up” proteomics studies.25 Trypsin cleaves
proteins at basic arginine and lysine residues and thus couples
Received: April 7, 2011
Accepted: May 24, 2011
Published: May 24, 2011
r
2011 American Chemical Society
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favorably with the affinity of crown ethers for protonated amine
moieties.26ꢀ28 We assess the utility of combining shift reagents
with IMS-IMS by examining peptides obtained upon tryptic
digestion of cytochrome c. Upon assessing the overall approach,
we illustrate an application of this method by the identification of
a peptide from an extremely complex mixture, human plasma.
The present work is closely related to a number of funda-
mental studies involving the binding of crown ethers to
amines.26ꢀ28 These systems have been useful models in the
study of molecular recognition,27ꢀ29 gas-phase hydrogen/
deuterium exchange,26 and the mobile proton model in the
context of collision-induced dissociation (CID) of peptide
ions.26 As described by others,28 the presence of 18C6 in the
electrospray solution is found to alter the ionization efficiency as
well as the charge state distributions of many peptide ions. A
recent report suggests that measurements of these complexes are
sensitive to molecular motion within supramolecular com-
plexes.30 We also discuss some features pertaining to the struc-
tures of peptideꢀcrown complexes.
was digested with trypsin using standard procedures described
previously.22 The lyophilized product was prepared for electro-
spray ionization (ESI) in solution (49:49:2 waterꢀacetonitrileꢀ
acetic acid) at a concentration of 0.25 mg mLꢀ1. Plasma samples
were prepared with abundant proteins depleted as previously
reported.18 The plasma peptides were dissolved in 49:49:2
waterꢀacetonitrileꢀacetic acid and brought to an estimated
total peptide concentration of ∼0.3 mg/mL. Noncovalent com-
plexes were formed by addition of 50 mol equiv of 18-crown-6
ether (99% purity, Aldrich, St. Louis, MO) to an aliquot of the
digest solutions. A syringe pump (kd Scientific, Holliston, MA)
was used to deliver sample at a flow rate of 0.25 μL minꢀ1
through a pulled capillary tip (75 μm i.d. ꢁ 360 μm o.d.) biased
2.2 kV above the drift voltage.
Cross Section Measurements. Drift times measured for
peptides and peptideꢀcrown complexes can be converted into
collision cross sections using the following equation38
ꢀ
ꢁ
1=2
ð18πÞ
ze
1
1
1=2tDE 760
ð1Þ
P 273:2 N
T
1
Ω ¼
þ
1=2
16
mI mB
L
ðkBTÞ
’ EXPERIMENTAL SECTION
where ze is the charge of the ion, kB is Boltzmann’s constant, and
T refers to the temperature of the buffer gas. Values mI and mB
correspond to the masses of the ion and buffer gas (in this case,
He atoms), respectively. The measured drift times are repre-
sented by tD, and parameters L, P, and N correspond to the
length of the drift separation, the buffer gas pressure, and the
buffer gas number density, respectively.
Database Search for Peptide Identifications. Peptides from
human plasma were assigned using database searches. Fragment
peak lists were compiled into text files and searched against the
Swis-Prot human database using a MASCOT software suite.39
To allow for the presence of the remaining 18C6 adducts, the
precursor mass used in the search was adjusted to account for
neutral losses of this species. As part of the MASCOT search, a
threshold score is generated to indicate the statistical relevance of
the matched results.
Instrumentation. General discussions of IMS experiments
and theory as well as IMS-MS combinations have been reported
elsewhere.1,16,31ꢀ35 Experiments reported here were conducted
on two similar home-built IMS-IMS-MS instruments differing
mainly in the total length of the drift tube. Early studies were
conducted on a drift tube roughly 2 m in length; later experi-
ments were conducted on a drift tube ∼3 m in length. Only a
short description of these instruments is provided here; a detailed
report has been published previously.16 Briefly, electrosprayed
ions are accumulated in a source ion funnel and are released in
150 μs-wide pulses into the drift tube where they diffuse through
a buffer gas (∼3.0 Torr He at 300 K) under the influence of a
weak uniform electric field (9 V cmꢀ1). For IMS-IMS experi-
ments, the total drift tube length is divided into two drift regions
separated by an electrostatic gate used to transmit mobility-
selected ions. The selection of these ions is defined by a variable
delay that is triggered by the source pulse of the drift experiment.
Selected ions are then heated through high-field collisions in an
ion activation region to produce a new distribution of ions prior
to entering the second drift region. The extent of activation can
be tuned by changing the voltage drop across the lenses (spaced
∼0.3 cm apart) comprising the activation region. A difference of
5 V is used for passive transmission of ions, whereas 200 V
difference or greater leads to substantial CID of peptide ions. In
these studies, activation voltages of 125 and 165 V were examined
for dissociation of noncovalent complexes. Upon exiting the drift
tube, the ions enter the source of an orthogonal reflectron time-
of-flight (TOF) mass spectrometer. For the types of ions
investigated here, we estimate this mass analyzer exhibits a
resolution of ∼2500 with a mass accuracy of ∼0.4 Da. Because
the time scale of TOF analysis is 3 orders of magnitude smaller
than that of the IMS analysis (tens of microseconds compared to
tens of milliseconds), roughly one thousand mass spectra are
collected over the course of a single drift separation. Nested
measurements of drift times (tD) and m/z are collected to
construct the IMS-MS data as described previously.36,37 We
report tD and m/z values using the tD(m/z) nomenclature
convention described elsewhere.36
’ RESULTS AND DISCUSSION
IMS-MS Data Sets for Peptides and PeptideꢀCrown Com-
plexes. Figure 1 shows the nested tD(m/z) distributions for the
tryptic peptides of cytochrome c and the corresponding
peptideꢀ18C6 mixture. Experimentally determined m/z values
and cross sections (converted from experimental drift times
using eq 1) are in agreement with those reported previously.40
A theoretical peak list for peptideꢀcrown complexes was con-
structed using the monoisotopic mass of 18C6 (264.16 Da), and
a summary of assignments is provided in Tables 1 and 2. The
nomenclature used for assignments is as follows. The first
number in the label (Figure 1 and Tables 1 and 2) corresponds
to the charge state of the ion. A letter is assigned to the peptide
sequence in alphabetical order with increasing peptide mass. For
peptideꢀcrown complexes, a second number is added to denote
the number of adducts on the ion. As described previously,41 ions
of the same charge state fall into families and are shown by the
white diagonal lines labeled [M þ H]þ, [M þ 2H]2þ, [M þ
3H]3þ, and [M þ 4H]4þ. Intense peaks observed at low m/z
have been assigned as [18C6 þ H]þ, [18C6 þ H2O þ H]þ,
[18C6 þ Na]þ, and [18C6 þ K]þ and appear at 10.9(265.2),
10.9(283.2), 10.5(287.1), and 11.0(303.3), respectively.
Sample Preparation and Electrospray Conditions. Cyto-
chrome c (horse heart, 90% purity from Sigma, St. Louis, MO)
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Figure 1. Two nested tD(m/z) plots for a tryptic digest of equine cytochrome c sprayed alone (a) and with 18C6 (b). Diagonal lines depicting the
general location of the observed charge states are labeled on each graph. The vertical line on part b shows the selections made in the experiments shown
in Figure 2. Assignments for peak labels can be found in Tables 1 and 2 for parts a and b of Figure 1, respectively. The first number corresponds to the
charge state, the letter was alphabetically assigned to the observed peptides in order of increasing neutral mass, and the second number distinguishes the
number of crown ether adducts associated with the peptide.
Table 1. Peptides Observed in IMS-MS Analysis of Cytochrome c Digest
peptide sequence
sequence labela
neutral massb
[M þ H]þc
[M þ 2H]2þc
[M þ 3H]3þc
GGKHK
a
b
c
d
e
f
525.30
603.34
GITWK
1b
1c
1d
1e
2b
2c
2d
2e
2f
IFVQK
633.38
YIPGTK
677.37
MIFAGIK
778.44
KYIPGTK
805.47
TGPNLHGLFGR
YIPGTKMIFAGIK
TGQAPGFTYTDANK
EETLMEYLENPK
KTGQAPGFTYTDANK
EETLMEYLENPKK
IFVQKCAQCHTVEK
g
h
i
1167.61
1437.80
1469.68
1494.69
1597.77
1622.79
1632.81
2g
2i
j
2j
k
1
m
2k
2l
3k
2m
3m
a Each peptide observed was assigned a letter alphabetically with increasing neutral mass for ease of use in discussion and labeled spectra. b Monoisotopic
neutral masses calculated using in silico digestion. c To specify a peak, the sequence label is preceded by a number corresponding to the charge state at
which it was observed.
Inspection of these data in Figure 1 shows that the spectrum for
the peptideꢀcrown mixture contains many more peaks compared
to the data for the peptide sample. Most of the additional peaks are
associated with variations in the binding stoichiometry between
18C6 and the peptides. For example, consider the peak 2g in
Figure 1 at tD(m/z) = 14.3(584.8). Upon addition of 18C6, the
same peptide is represented by peaks 3g1, 3g2, 3g3, and 3g4,
present at 12.6(478.3), 14.4(566.3), 15.0(654.4), and 16.2(742.4),
respectively. This is a general feature of the system and results in
each peak in Figure 1a splitting into multiple peaks in Figure 1b.
Some additional peaks are associated with peptide sequences that
are detected exclusively in the sample with 18C6. These sequences
(GGKHK and YIPGTKMIFAGIK) correspond to missed clea-
vages and are presumably present at a lower abundance in solution.
It seems likely that the increased electrospray ionization efficiency
afforded by complexation with 18C6 (as observed previously by us
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Table 2. Peptides Observed in IMS-IMS-MS Analysis of Cytochrome c Digest Using Shift Reagents
peptide sequence
neutral mass
charge states
values of n
GGKHK
525.30
603.34
633.38
677.37
778.44
805.47
1167.61
1437.80
1469.68
1494.69
[M þ n(18C6) þ 2H]2þ
[M þ n(18C6) þ 2H]2þ
[M þ n(18C6) þ 2H]2þ
[M þ n(18C6) þ 2H)2þ
[M þ n(18C6) þ 2H]2þ
[M þ n(18C6) þ 2H]2þ
[M þ n(18C6) þ 3H]3þ
[M þ n(18C6) þ 3H]3þ
[M þ n(18C6) þ 2H]2þ
[M þ n(18C6) þ 2H|2þ
[M þ n(18C6) þ 3H]3þ
[M þ n(18C6) þ 3H]3þ
[M þ n(18C6) þ 3H]3þ
[M þ n(18C6) þ 2H]2þ
[M þ n(18C6) þ 3H]3þ
[M þ n(18C6) þ 4H]4þ
1ꢀ3 (2a1ꢀ2a3)a
0ꢀ3 (2b0ꢀ2b3)
0ꢀ3 (2c0ꢀ2c3)
0ꢀ3 (2d0ꢀ2d3)
1ꢀ3 (2e1ꢀ2e3)
1ꢀ3 (2f1ꢀ2f3)
1ꢀ4 (3g1ꢀ3g4)
1 (3h1)
GITWK
IFVQK
YIPGTK
MIFAGIK
KYIPGTK
TGPNLHGLFGR
YIPGTKMIFAGIK
TGQAPGFTYTDANK
EETLMEYLENPK
1 (2i1)
0ꢀ1 (2j0ꢀ2j1)
1ꢀ2 (3j1ꢀ3j2)
0ꢀ4 (3k0ꢀ3k4)
1ꢀ4 (3l1ꢀ3l4)
1ꢀ2 (2m1ꢀ2m2)
1ꢀ4 (3m1ꢀ3m4)
1ꢀ2 (4m1ꢀ4m2)
KTGQAPGFTYTDANK
EETLMEYLENPKK
1597.77
1622.79
1632.81
IFVQKCAQCHTVEK
a Peak labels follow the same notation as Table 1 with one addition; the number at the end of the label denotes the number of 18C6 adducts complexed to
the ion.
Figure 2. A selected packet of ions with total drift times in the range of 15.52ꢀ15.73 ms is transmitted with no activation (left panel), with activation at
125 (middle panel) and 165 V (right panel).
and others)28 is responsible for the detection of these peptides.
This feature of the complexation analysis is potentially useful in
proteomic analyses, where detection of multiple peptides is often
required for identification of a protein.42
Another feature of the complexation analysis with 18C6 is
observation of ions at higher charge states. While the peptides
identified from the typical ESI solution produced ions with z =
1ꢀ3, peptideꢀcrown complex ions are found with z = 2ꢀ4. The
charge increase results from stabilization of the charged sites.28
Additionally, higher charge states may be facilitated by dielectric
shielding of the charged site upon binding 18C6. It is worth
noting that peaks corresponding to complexes are similar in
width to those without 18C6 adducts. It appears that peaks do
not broaden upon complex formation.
IMS-IMS Analysis of PeptideꢀCrown Complexes. Figure 2
shows a blown up region of the nested data sets corresponding to
a mobility selection that leads to a total drift time at the vertical
dashed line in Figure 1 (15.52ꢀ15.72 ms total drift time).
Additionally, plots of data recorded at two collisional activation
voltages (125 and 165 V) are shown. The spectrum for the
selection (left panel) is dominated by peaks 3m3, 2d3, 2c3, 2d2,
and 2c2. Collisional activation of the selected ions at 125 V,
shown in Figure 2 (middle), is generally sufficient to remove at
least one 18C6 adduct from the complexes, generating the peaks
3m2 and 2c1 at 15.2(721.4) and 14.4(449.8) as the major
products. At higher voltages (165 V, Figure 2, right) a greater
extent of dissociation is observed and some portion of the bare
peptides are recovered. Evidence for this is found from peak 2c0
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Figure 3. Effect of peptide or peptideꢀcrown complex on shift magnitude afforded by multiple additions of 18C6. Cross section of the bare peptide is
shown on the x-axis, and the increase in cross section upon complexation is shown on the y-axis. Data points that fall on a vertical line correspond to the
same peptide ion from the cytochrome c digest. Single, double, triple, and quadruple additions of 18C6 are shown by diamonds, squares, triangles, and
exes, respectively. Solid data points denote doubly charged ions, and open data points denote triply charged ions.
that is resolved in drift time at 12.9(317.7). This activation was
also sufficient to produce some peptide fragment ions, shown by
the labeled diagonal line. Evidence for charge reduction is also
found. In this case, the small 2m0 peak corresponding to
[IFVQKCAQCHTVEK þ 2H]2þ ion is likely produced from
peaks 3m0 or 3m1 corresponding to [IFVQKCAQCHTVEK þ
3H]3þ and [IFVQKCAQCHTVEK þ 18C6 þ 3H]3þ, respec-
tively. The [18C6 þ H]þ ion can be observed in some of the
activated data sets (data not shown), suggesting that abstraction
of the proton by the 18C6 adduct contributes to the charge
transfer process.
Influence of Complexation on Cross Section. It is interest-
ing to compare the cross sections of the peptideꢀcrown com-
plexes to those of the naked peptide ions. This can be done prior
to complex formation and upon dissociation. Drift time distribu-
tions for some peptide ions are similar in appearance when
compared to their complexed forms, with only a constant offset
in total drift time. For example, the distribution for the peptide
ion 3 m remains similar in its pattern for all peptideꢀcrown
stoichiometries observed. In these cases, it is tempting to assume
that the backbone conformation of the peptide ion remains the
same and the 18C6 ligands simply cap charged sites without
structural distortion. In other examples, however, the drift time
distributions change drastically (e.g., peptides that appear as
singlets form multiplets upon complexation or vice versa). For
example, although it appears predominantly as a single feature in
the peptide-only sample, [KTGQAPGFTYTDANK þ 3H]3þ
(peak 3k) appears as a doublet in its [KTGQAPGFTYTDANK
þ 18C6 þ 3H]3þ form (peak 3k1). In contrast, [TGQAPGF-
TYTDANK þ 2H]2þ (peak 2i) appears to adopt a narrower
distribution of structures upon binding a single 18C6 adduct
(peak 2i1). As such, there is very little that can be stated gener-
ally about the effect of 18C6 complexation on peptide struc-
ture; rather, the complexed form of a peptide ion appears to be
sequence dependent in a way that is not straightforward to
anticipate.
illustrates the effects of peptide size, degree of complexation,
and charge state on the overall cross section of peptideꢀcrown
complexes. In this plot, a vertical line of points corresponds to the
same peptide ion and the spacing between points represents the
increasing cross section of a growing complex. Additionally,
the effect of a given number of 18C6 adducts can be observed
as the size of the peptide varies. As expected, larger peptides
experience smaller increases in cross section upon complexation
with 18C6 because the adduct comprises less of the total size of
the complex for larger peptides. From Figure 3, we estimate the
increase in size associated with incorporation of a crown on a
peptide to be ∼20ꢀ40 Å2.
It is interesting to compare cross sections of peptide ions
formed directly by ESI and subsequently activated with those
that arise upon dissociation of the peptideꢀcrown complex.
Overall, these cross sections are similar. This observation is
consistent with studies of peptide conformer distributions that
are produced upon collisional activation of isolated confor-
mers.43 It appears that, after dissociation, the activated ions
continue to anneal, and the peptides form a relatively stable
distribution of structures.
Assessing the Value of Complexation for Separation
Capacity. Although addition of 18C6 clearly benefits IMS-IMS
capabilities, the addition of this reagent also leads to a far more
complex sample and more complicated data sets. Here we assess
the enhancement gained compared to the cost of additional
complexity. One means of quantifying this complexity is to
compare the total number of peaks observed for a particular
peptide sequence to the number observed for the peptideꢀ
crown complexes. We include different charge states and differ-
ent numbers of 18C6 adducts, as each will appear as a unique
peak. The ratio of these two numbers is the factor by which
addition of 18C6 increases the complexity of the data set.
As an example, consider the MIFAGIK peptide. This peptide
is observed as peaks 1e and 2e without addition of 18C6. In the
presence of 18C6, three peaks from MIFAGIK were identified
(2e1, 2e2, and 2e3). Additionally, 2e1 was present in a selection
made in an IMS-IMS-MS experiment and produced the bare
peptide (2e0). Thus, in total, four peaks related to the sequence
Comparison of Complexes with Different Numbers of
Crowns. Some trends are apparent upon comparison of peptideꢀ
crown complexes of different sizes. The data in Figure 3
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resolved. In addition to ensuring a shift occurs, the loss of 18C6
induces a shift that is larger than is typically associated with
different peptide conformers.
Analysis of the peak widths, and the range over which peaks are
observed, allows us to estimate the analytical peak capacity.
Whereas the maximum observed shift in an IMS-IMS analysis of
tryptic peptides was (11%,22 loss of a single 18C6 adduct shifts
the peak in total drift time by ∼20ꢀ25%. Increased shifts in the
second IMS region directly lead to higher two-dimensional peak
capacity.44 IMS-IMS analysis of [MIFAGIK þ n(18C6) þ
2H]2þ ions (n = 1ꢀ3) gave rise to peaks with an average full-
width-at-half-maximum (fwhm) value of 0.255 ms. These peaks
are observed to extend over a range of 7.75 ms. Thus, we calculate
a peak capacity of ∼30 (7.75 ms/0.255 ms). Peaks associated
with the [IFVQKCAQCHTVEK þ n(18C6) þ 3H]3þ ions (n =
1ꢀ4) had an average fwhm value of 0.233 ms and occupied a
range of 6.88 ms, also resulting in a peak capacity of ∼30. The
peak capacity of the initial drift separation is between 60 and 80
for tryptic peptide ions.22 Therefore, the data reported here
suggest a maximum total IMS-IMS peak capacity of between
1800 and 2400 when shift reagents are used. This is substantially
greater than the 480 to 1360 peak capacity range determined for
IMS-IMS without 18C6 shift reagents.22
Analysis of Human Plasma Peptides with 18C6 Shift
Reagent. With the added advantages in peak capacity and signal
redundancy, it is interesting to apply the IMS-IMS technique to a
system of extraordinary complexity. Our aim was to examine
whether the IMS-IMS technique combined with 18C6 shift
reagents could simplify a complex system to the point where
distinct components could be isolated and identified by the single
stage of mass spectrometry.
Figure 4. Total drift time distributions for two sequences of cyto-
chrome c as analyzed by IMS-IMS (a). Same sequences analyzed by
IMS-IMS with 18C6 shift reagent (b).
MIFAGIK are observed in the 18C6 spectrum. We conclude that
the complexity for MIFAGIK is increased by a factor of 2.
Extending this analysis to all peptide assignments allows us to
estimate the average increase in complexity for all peptides in the
digest. We find 17 assignments in IMS-MS analysis of peptides
only, compared to 59 assignments in the peptideꢀcrown mix-
ture, an average complexity increase of a factor of ∼3.5.
Figure 5 shows typical results for the analysis of a human
plasma digest using IMS-IMS-MS with shift reagents. The
proteome of human plasma is remarkable. It is believed that at
any given time, on the order of 105 proteins may be present.
Thus, we anticipate that more than a million peptide signals may
exist. The largest signals should be associated with peptides from
∼40 abundant proteins that are known as the classical plasma
proteins.45 We (and others) have characterized plasma proteins
from their tryptic peptides using a range of liquid chromatogra-
phy and MS techniques.42,46ꢀ48 Our findings (from a multi-
dimensional separation involving strong-cation exchange and
reversed phase liquid chromatography, coupled with IMS-MS
detection) provided high-confidence identification of 2928 pro-
teins (and many more signatures for species at lower confidence
levels).42 In direct IMS-MS analysis of tryptic peptides and
peptideꢀ18C6 mixtures (with no chromatography), spectra
such as those shown in Figure 5 may contain ∼104ꢀ105 features
(and many distinct peaks).42 A comparison of the IMS-MS
results with MS-only (top panels of Figure 5) shows that many
peptideꢀ18C6 peaks that are buried under the baseline signal in
the MS-only spectrum are apparent when the IMS separation is
employed.
When ions are allowed to separate in the first IMS region and an
activation gate is used to select narrow regions for further
separation, the spectra are dramatically simplified. An example of
this is also shown in Figure 5, for mobility-selection of a narrow
distribution of ions having a total drift time of ∼32 ms (second
panel). The mass spectrum that accompanies this selection
indicates that many abundant peaks are present in this narrow
drift time. Upon activation of these ions (using 165 V), the
complexes dissociate. The resulting IMS-MS plot shows the
These redundant peaks increase sample complexity but can
provide valuable information. In cases where complexes appear at
low abundance, or in a congested region of the spectrum, the
increased complexity provides opportunities to detect (or select)
ions. Finally, unique peaks from analyte ions that are only
detected when electrosprayed with 18C6 add to the estimate
of complexity above but provide valuable information that is
otherwise absent.
An example of a favorable scenario is shown in Figure 4. IMS-
IMS distributions for two peptide ions from cytochrome c digest,
[MIFAGIK þ 2H]2þ and [IFVQKCAQCHTVEK þ 3H]3þ, are
shown. This plot corresponds to ions that were selected at 25.1
and 25.3 ms, respectively, and subsequently activated at 165 V.
Although some of the [IFVQKCAQCHTVEK þ 3H]3þ ion
population shifts to lower mobility, the majority remains close to
the mobility at which it was selected. Thus, the IMS-IMS sep-
aration does not change the mobilities of these ions substantially,
and significant overlap between the [IFQVKCAQCHTVEK þ
3H]3þ and [MIFAGIK þ 2H]2þ peaks remains. Figure 4
also shows an analogous experiment using 18C6 shift re-
agent. The precursors for this IMS-IMS separation were the
[IFQVKCAQCHTVEK þ 2(18C6) þ 3H]3þ and [MIFAGIK
þ 2(18C6) þ 2H]2þ ions, which were selected at 34 and 38 ms
and activated to yield the [IFQVKCAQCHTVEK þ 18C6 þ
3H]3þ and [MIFAGIK þ 18C6 þ 2H]2þ ions. The shift in
mobility observed upon 18C6 loss allows these species to be
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Figure 5. The top panel features an IMS-MS nested plot of human plasma peptides electrosprayed from a solution containing 18C6 with the total mass
spectrum to the right. Mobility-selected complex ions (second panel) are activated with 165 V to dissociate the complex ions into bare peptide ions,
which resolve further in mobility (third panel). Integration of the data between the dashed lines shows an isolated precursor in the drift dimension (third
panel, right). Parallel dissociation at the end of the drift tube renders mobility-labeled fragments (fourth panel). Integration between the dashed lines
now provides fragmentation spectrum that matches [KVPQVSTPTLVEVSR þ 3H]3þ (fourth panel, right). The ion score for this match was 43, well
above the conservative statistical relevance threshold of 30.
advantage of the complexation approach. Large shifts in the m/z
values and mobilities are observed. The smaller complexes and
naked peptides lead to signals that are dispersed from ∼24 to
32 ms. Such a large shift makes it possible to find regions that are
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dominated by a single peak (or a few peaks). A selection of a
narrow region at 32 ms allows us to integrate a mass spectrum that
is dominated by a peak at m/z = 635.3. Although other IMS times
show similar simplification, we focus on this peak for assignment.
Our IMS-IMS-MS instrument is equipped with an activation
region in the middle of the drift tube as well as one at the exit
(as described previously).16 When a set of mobility-separated
ions is dissociated at the exit of the drift tube, we can align
fragment peaks with their antecedent precursors (because the
fragments and precursors are coincident in drift time). We refer
to this as parallel dissociation.49
The final spectra shown in Figure 5 (bottom) correspond to
parallel dissociation of precursors at the exit of the drift tube.
These ions originated from the ESI source as peptide/18C6
complexes and were separated in an initial IMS region. A subset
of these complexes was selected after the first IMS region,
activated to dissociate the complexes, and then allowed to
separate again prior to dissociation. In the present case we show
dissociation of the precursor ion (that after these steps of
separation is relatively isolated) at m/z = 635.3. The fragmenta-
tion spectrum for the isolated m/z = 635.3 ion can be obtained by
integrating the signal at 32 ms. Upon activation, we find a large
peak at m/z = 547.3, 88.0 Th less than the precursor state. This
88.0 Th difference is a signature of the loss of 18C6 from a triply
charged peptide. An inspection of the isotopic spacing of the
m/z = 547.3 peak confirms that this peak corresponds to a triply
charged ion. The overwhelming majority of the remaining peaks
correspond to singly charged species; there are no additional
88 Th differences, which if observed, would suggest that the
m/z = 547.3 peak includes additional 18C6 molecules.
Thus, we begin interpreting this fragmentation pattern by
assuming that the peak at m/z = 547.3 corresponds to a triply
charged peptide ion precursor. Using this value and a list of peaks
present in the spectrum as input for a database search, we find
that our spectrum is likely due to the [KVPQVSTPTLVEVSR þ
3H]3þ peptide. This peptide is often detected in our plasma
studies, as it arises from digestion of the most abundant protein in
plasma, albumin. That we have identified this peptide directly
with a single MS analysis suggests that the present approach has
substantial utility. Other regions of the spectra show additional
peptides from albumin as well as other proteins.1 At this point in
the analysis we have not characterized the range of protein
abundances that can be detected directly with this approach.
However, such an analysis requires only a few minutes to record
and the early results appear promising.
’ SUMMARY AND CONCLUSIONS
We have examined IMS-IMS-MS analyses of tryptic peptides
bound to 18C6 as a shift reagent. In this approach, electrosprayed
peptideꢀcrown complexes resolve in the first IMS dimension. A
narrow distribution of complexes is mobility-selected and sub-
sequently dissociated to yield a new distribution (including
smaller complexes, naked peptides, charge transfer products,
and fragments) which disperse in a second mobility separation.
Analysis of a tryptic digest of cytochrome c using this approach
shows increased IMS-IMS peak capacity (up to ∼2400) and
detects two additional peptides compared to IMS-IMS alone.
The peptides identified exclusively from the peptideꢀcrown
mixture correspond to sequences with missed cleavages and
are presumably at lower abundance than the tryptic peptides.
An example of an analysis aimed at identifying a peptide from a
complex mixture of peptides generated by digestion of human
plasma was described. In this approach it was shown that this
combination of techniques was sufficient to isolate an individual
peptide from a direct-infusion electrospray ion source; and
dissociation of the isolated peptide matched a peptide associated
with an abundant protein in plasma.
While the ability to dissociate species increases the total IMS-
IMS peak capacity (compared with only structural changes), the
addition of 18C6 introduces a substantial increase in the com-
plexity of the samples to be analyzed. This occurs because of the
range of peptideꢀ18C6 stochiometries and charge states, leading
to multiple peaks for each peptide. On average, we estimate
spectra to be a factor of ∼3.5 times more complex than those for
peptides in the absence of 18C6. We note that only a few
favorable cases (such as those highlighted here) may be needed
to appreciably increase the information content of an analysis.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: clemmer@indiana.edu.
’ ACKNOWLEDGMENT
We acknowledge Amy Hilderbrand, Stormy Koeniger, and
Samuel Merenbloom for helpful discussions during early stages
of this project. This research was supported in part by the Indiana
METACyt Initiative of Indiana University and a grant from the
National Institutes of Health (Grant 1RC1GM090797-01).
Finally, it is interesting to note that peptideꢀ18C6 complexes
can be used directly for fragmentation studies to identify peptide
sequences. Moreover, these methods may have merit in helping
to define the charge states of precursor ions, when only low-
resolution MS data are available.50 As reported in previous CID
studies on peptideꢀcrown complexes,26 18C6 sequesters the
charge on a protonation site and inhibits dissociation pathways
involving a mobile proton.51 The result of this is that the 18C6
adduct must dissociate before the proton can become mobile and
induce substantial peptide bond breakage. Because 18C6 must be
eliminated prior to peptide dissociation, fragmentation of a
peptideꢀcrown complex is probably less efficient compared to
the corresponding naked peptide ion under identical conditions.
Nonetheless, it appears that partially dissociated complexes can
serve as precursor ions for fragmentation, should any of the
naked peptide be recovered at low abundance or fail to isolate
sufficiently in mobility.
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