Review

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Developing liquid chromatography

ion mobility mass spectometry
techniques
Stephen J Valentine, Xiaoyun Liu, Manolo D Plasencia,
Amy E Hilderbrand, Ruwan T Kurulugama, Stormy L Koeniger
and David E Clemmer†
When a packet of ions in a buffer gas is exposed to a weak electric field, the ions will
separate according to differences in their mobilities through the gas. This separation forms
the basis of the analytical method known as ion mobility spectroscopy and is highly
CONTENTS efficient, in that it can be carried out in a very short time frame (micro- to milliseconds).
Analytical challenge Recently, efforts have been made to couple the approach with liquid-phase separations
associated with complex and mass spectrometry in order to create a high-throughput and high-coverage
mixtures of proteins approach for analyzing complex mixtures. This article reviews recent work to develop this
Fundamentals & approach for proteomics analyses. The instrumentation is described briefly. Several
instrumentation multidimensional data sets obtained upon analyzing complex mixtures are shown in
1D-LC-IMS-MS order to illustrate the approach as well as provide a view of the limitations and required
measurements future work.
2D-LC-IMS-MS
Expert Rev. Proteomics 2(4), 553–565 (2005)
measurements
Current limitations Analytical challenge associated with instrument time. Even more daunting is that
Future directions complex mixtures of proteins protein concentrations in plasma differ by
Conclusions Consider 1 ml of human plasma. It is believed more than ten orders of magnitude (from
that such a sample could contain thousands of ∼10 mg/ml to <1 pg/ml) and some are tempo-
Expert commentary
& five-year view
different proteins in measurable quantities [1]. rally dependent, varying with diet, age, state
The ability to characterize them all would pro- of health and so on [1]. With this in mind, it is
Key issues
duce physiologically relevant signatures and perhaps not surprising that the number of
References has become a significant challenge for an novel proteins that can be used to characterize
Affiliations emerging group of biotechnologists who aim physiologic state (i.e., biomarkers) remains rel-
to understand the molecular mechanisms of atively low [13,14].
diseased states [2–12]. However, the analytical
issues that are associated with analyzing Overview of this review
plasma in detail are difficult to overstate. For In this paper, recent work aimed at developing
example, proteolytic digestion of a mixture of a high-throughput and high-coverage approach
†
Author for correspondence 104 proteins in which an average of 50 frag- for analyzing complex mixtures such as
Indiana University, Department of
Chemistry, 800 E. Kirkwood Ave.
ments for each protein were produced would plasma is reviewed. The approach is based on
Bloomington, IN 47405, USA lead to a mixture of approximately 5 × 105 a combination of liquid chromatography (LC)
Tel.: +1 812 855 8259 peptides. An ideal separation of this mixture, with ion mobility spectrometry (IMS) [15]
Fax: +1 812 855 8300 in which peptides line up and exit the chroma- and MS. The mobility of an ion through a
clemmer@indiana.edu tographic column at a rate of one per second buffer gas depends upon the ion’s charge and
KEYWORDS: (near the maximum rate for mass spectro- collision cross-section with the buffer gas.
complex mixture, metry [MS]-based detection and identification Since gas densities are much lower than the
ion mobility spectrometry, liquid
chromatography, LC-IMS-MS, approaches, i.e., MS and tandem MS density of the condensed phase, the separa-
mass spectrometry, plasma [MS/MS]) would require nearly 1 month of tion can be carried out on very short time

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Valentine, Liu, Plasencia et al.

scales (usually in the order of milliseconds). Thus, it is possible same fashion with condensed-phase separations to provide
to include the mobility separation within a LC and MS analysis three dimensions of component dispersion [19]. Since this
to create a 3D-LC-IMS-MS experiment. Here, the following experiment, the multiply dispersive approaches (LC-IMS and
topics are covered: LC-IMS-MS) have been used to analyze mixtures of small mole-
• The background of IMS separations and the basics of cules, tryptic peptides, combinatorial libraries and proteome
LC-IMS-MS instrumentation digests [19–24].
Multidimensional LC-IMS-MS data sets are acquired by
• Example data sets for several selected complex systems,
nesting rapid analyses within those requiring longer times
including human plasma
[18,19]. That is, if the time scales of the multiple separations are
• A discussion of current limitations and future improvements substantially different (minutes, milliseconds and micro-
At this point, LC-IMS-MS is an emerging technology, and seconds), then each separation can be carried out within indi-
although it appears that it offers significant advantages com- vidual time windows of the preceding step. For example, within
pared with LC/MS alone, much work is required before it will a 20 ms window used to record the IMS separation, one could
be used routinely – perhaps not unlike the state of high-resolu- acquire hundreds to thousands of flight time distributions con-
tion Fourier transform MS and high-throughput LC/MS/MS stituting the entire mass spectrum of the mobility-dispersed
approaches a decade ago. ions. Such an approach results in a 3D dispersion of each mix-
ture component such as a peptide from a proteomic digest sam-
Early developments associated with multidimensional ple. The hypothetical plots shown in FIGURE 1 illustrate a partic-
techniques that incorporate IMS ularly useful configuration. If ions are transmitted through the
Early in its development, IMS was coupled to various separa- drift tube gently (FIGURE 1A), then the dispersion results in the
tion strategies including gas chromatography (GC), supercriti- indicated positions of the precursor ions. On the other hand, if
cal fluid chromatography (SFC) and LC [16]. Although such ions are activated between the IMS and MS separation steps
work utilized two separation steps, it did not make full use of (FIGURE 1B), then a fragmentation spectrum can be recorded.
both dimensions of separation because limitations in electro- This approach has been used to generate MS and MS/MS
nics required that at least one dimension of the system be oper- information for ions in parallel [25].
ated in a scanning rather than dispersive mode [17]. In the late Data generated by LC-IMS-MS measurements are described
1990s, advances in electronics and data acquisition systems using a standard nomenclature developed recently [18].
enabled the development of the first multiply dispersive Reported time values for each dispersive measurement are
method, IMS coupled with time-of-flight (TOF)-MS [18]. bracketed in their nesting order [18,19]. For example, experimen-
Shortly thereafter, IMS-MS measurements were coupled in the tal time values (FIGURE 1) for data obtained from a LC-IMS-
TOF analysis would be listed as tR[tD(tF)],
where tR, tD and tF correspond to the
retention time, drift time and flight time
A B of each species, respectively. Often, the
tF = µs tF = µs ions’ tF is converted to a mass-to-charge
ratio (m/z) using a standard, multipoint
calibration. For experiments that utilize
two dimensions of LC (e.g., those that
couple strong cation exchange [SCX] with
LC), the first-dimension elution time
(e.g., tSCX) or fraction number can also be
included in the nomenclature. Thus, a
tR = min tR = min 2D-LC-IMS-MS measurement may
report values for data set features as
tSCX{tR[tD(tF)]} [26].

Fundamentals & instrumentation

tD = ms tD = ms Mobility measurements
Several excellent reviews discuss the funda-
Precursor CID mentals associated with mobility measure-
ments in detail [27–31]; only a brief descrip-
Figure 1. Hypothetical plots of data obtained from LC-IMS-MS (A) and LC-IMS-MS/MS (B) tion of IMS as it pertains to a multiply
experiments. See text for details. dispersive approach is provided here. The
CID: Collision-induced dissociation; IMS: Ion mobility spectrometry; LC: Liquid chromatography;
MS: Mass spectrometry; MS/MS: Tandem mass spectrometry.
time required for an ion to traverse a
region filled with an inert buffer gas under

554 Expert Rev. Proteomics 2(4), (2005)


Ion optics
ESI source
Pulled-tip
Linear
RPLC nanocolumn
ion trap
cap pump
Figure 2. Schematic diagram of instrumentation used in LC-IMS-MS studies.
ESI: Electrospray ionization; IMS: Ion mobility spectrometry; LC: Liquid chromatography; MCP: Microchannel plate; MS: Mass spectrometry; RPLC: Reverse-phase
liquid chromatography; TOF: Time-of-flight.
the influence of a weak electric field is related to the mobility (K)
of the ion [32]. Under low-field conditions, the mobility of an ion
through the buffer gas is given as K = vD.E-1 (vD is the drift veloc-
ity of the ion and E is the electric field) [32]. Often, to permit
comparison between different measurements, ion mobilities are
reported as reduced mobilities (K0) calculated from [32]:
2 that favor transmission of precursor ions (low-field conditions)
L 273.2 P
K 0 = ------------- × ------------- × ---------
tD ⋅ V T 760
Here tD, L, V, P and T correspond to the measured drift time,
length of the drift region, the applied drift voltage, and the
pressure and temperature of the buffer gas, respectively. IMS
measurements are extremely reproducible and any two
measurements from the same instrument typically agree to
within approximately 1–2%. Ions that adopt compact confor-
mations have higher mobilities than those that exist as extended
conformers [28,29]. For ions with similar collision cross-sections,
those that exist as higher charge states have higher mobilities as
they are influenced by a greater drift force [28,29,33,34].
LC-IMS-MS instrumentation
A schematic diagram of one existing LC-IMS-MS instrument
constructed to analyze complex mixtures is shown in FIGURE 2.
Here, mixture components are separated with LC on a
reversed phase C18 column (75 µm × 15 cm). The column has
a pulled tip such that eluting species are electrosprayed into
the desolvation region of an electrospray ionization (ESI)
source. Ions are extracted from the high-pressure source into a
high-vacuum region and are focused into an octopole linear
ion trap (3D traps [35] and an electrodynamic ion funnel [36]
similar to that developed by Smith and coworkers [37,38] have
also been used). In the trap, ions are accumulated, stored and
are periodically pulsed into the drift tube. The drift tube
employs a split-field design that utilizes two field regions and
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Developing LC-ion mobility-MS techniques
First-field Second-field
region region TOF mass
spectrometer
Drift tube
Differential MCP/collision
pumping dynode detector
is filled with approximately 2.00 Torr He buffer gas [39]. The
first field region is approximately 20 cm long and contains
equally spaced electrostatic lenses to generate a uniform field
(∼5 V.cm-1). Ions are separated in this region according to their
mobilities (see above). The second field region of the drift tube
is approximately 1.2 cm long and is operated under conditions
or those that favor precursor fragmentation (high-field condi-
tions). During the course of a LC experiment, this field can be
modulated between conditions that favor transmission and
those that induce fragmentation of mobility-dispersed precursor
ions [40]. All ions exiting the drift tube are then focused into the
source region of a TOF mass spectrometer for mass analysis.
1D-LC-IMS-MS measurements
Urinary proteome
One of the first proteomics experiments to be performed using
a single dimension (1D) of LC coupled with IMS-MS tech-
niques was the study of a tryptic digest of the human urinary
proteome [22]. For these experiments, an instrument that incor-
porated an octopole collision cell after the drift separation and
before the mass analysis was used. Here, two separate LC runs
were recorded. The first and second runs utilized collision cell
voltages that favored transmission and fragmentation of precur-
sor ions, respectively. Upon completion of the experimental
runs, peaks from the fragmentation data set were linked to fea-
tures from the parent ion data set using tR and tD delineators.
This MS/MS information was then used to search a protein
database to obtain peptide identifications for features observed
in the precursor multidimensional data set.
FIGURE 3 shows a 2D tR(tD) plot of the human urinary pro-
teome digest. The data set demonstrates the utility of the gas-
phase mobility dispersion; many features that are not resolved
along the LC dimension are resolved along the mobility
555
Valentine, Liu, Plasencia et al.
dimension. Also shown is the base peak ion chromatogram. It
is estimated that a 2D peak capacity of approximately
6000–11,000 was achieved for this experiment. FIGURE 4 shows
a 2D tD(m/z) plot from the urinary proteome analysis obtained
using conditions that favored precursor fragmentation after the
mobility separation. As aforementioned, related fragments
and their precursors have the same drift times, thereby allowing
their grouping into MS/MS data sets [25]. Also shown in
FIGURE 4 are mass spectra obtained by integrating all bins within
three narrow drift time ranges. Identifications for tryptic pep-
tides corresponding to the proteins xanthine dehydroge-
nase/oxidase, uromodulin and serum albumin were obtained. A
preliminary analysis of the most intense features in the data set
yielded 27 peptide assignments corresponding to 13 proteins
(FIGURE 3). Although the preliminary work yielded few assign-
ments (largely a result of the extant informatics tools), it was
one of the first demonstrations of a proteomics analysis using
LC-IMS-MS techniques.
Drosophila melanogaster proteome
Other experiments utilizing a single LC separation step coupled
with IMS-MS techniques involved studies of the model organ-
ism Drosophila melanogaster. The first of these compared results
obtained from a proteomics analysis of the heads of three
Relative intensity
Retention time (min)
20 30 40 50 60
Human urinary proteome
8.07
Drift time (ms)
5.88
3.69
120 240 360 480 600 720
Frame number
Figure 3. Analysis of tryptic peptides from human urinary proteins.
The top plot shows the base peak chromatogram obtained from the
multidimensional (nanoflow LC-IMS-TOF) analysis of tryptic peptides
(0.83 µg) obtained from human urinary proteins. The data are plotted over
a retention time range of 18—92 min. The 2D tR(tD) plot at the bottom is
obtained by summing each tr and tD delineated mass spectrum and shows
the 2D separation resulting from the combined LC-IMS approach.
Reproduced with permission from [22], © 2003 American Chemical Society.
IMS: Ion mobility spectrometry; LC: Liquid chromatography;
TOF: Time-of-flight.
556
different organisms [26]. In all, more than 2.5 × 104 features
corresponding to protonated peptides were observed. Peak
intensities were compared for features identified in all three
samples to determine differences in individual protein expres-
sion. No statistically significant differences for the most
intense features were observed, suggesting that low-abundance
features (not identified) may to a large extent contribute to
individual characteristics.
Separate, ongoing experiments involving D. melanogaster
include the characterization of the proteome as a function of
embryogenesis (12 time points) [39]. These experiments were
performed on an IMS-MS instrument that has been slightly
modified from the version shown in FIGURE 2. To improve ion
storage and ESI source transmission capabilities, an electro-
dynamic ion funnel was incorporated at the front of the drift
tube, replacing the linear trap [36,38]. In this instrumental set-
up, ions accumulate at the back of the ion funnel and are
periodically pulsed into the drift tube to initiate mobility meas-
urements. Overall signal levels are increased by factors of 10–20
using the new instrumentation.
FIGURE 5 shows 2D tR(tD) plots of the data collected for an
embryo proteome digest at 0–2 h of development. The plots
demonstrate the complexity of the samples. Although the
drift dimension provides an increase in overall peak capacity,
FIGURE 5A shows that many features are indistinguishable
when plotting the entire data set, even when employing a
large intensity threshold. Typically, data are plotted as 2D
base peak plots where only the most abundant features at
each tR and tD are represented (FIGURE 5B). For these experi-
ments, the estimated peak capacity for the LC-IMS separa-
tion is approximately 6000. The overall number of proteins
observed in the various time point samples as well as changes in
protein expression as a function of embryogenesis are currently
70 80 90 under investigation.
2D-LC-IMS-MS measurements
Experimental set-up
Due to the gas-phase, mobility separation is a post-ionization
event, and it is possible to couple any number of condensed-
phase separations steps with IMS-MS analyses without sacri-
ficing ion signals. Thus, benefits in overall peak capacity and
component resolution associated with multiple dimensions of
LC can be availed by IMS-MS techniques. Several 2D-LC
strategies have recently been combined with IMS-MS meas-
840 960
urements. The first and more developed of these involves the
use of offline fractionation using SCX chromatography with
reversed phase LC-IMS-MS analysis. Although not described
here, it is noted that several other methods employing multi-
dimensional condensed-phase separations coupled with IMS-
MS are under investigation. Such experiments include the use
of size-exclusion and affinity chromatography techniques to
increase the resolution and sensitivity of the multiply disper-
sive measurements. Here, recent developments in studies uti-
lizing an SCX-LC-IMS-MS approach for proteomics analysis
are described.
Expert Rev. Proteomics 2(4), (2005)
 Developing LC-ion mobility-MS techniques

D. melanogaster mapping studies

The proteomes of two developmental A b2+
stages of D. melanogaster (adult head A B C 400 3 Xanthine
dehydrogenase/oxidase
and embryo) have been analyzed using 1500

Intensity
RMGGGFGGK
2D-LC-IMS-MS methods [41]. Experi- 200 y1 y3
y 5 (tentative)
mental data were compiled into high- 1300 a1 y2
y4
y7
dimension proteome maps containing 0
1100 y6
the tSCX, tR, tD and m/z values for each B b /y Uromodulin

Intensity
2+ 5 4
feature observed in the data sets, as well 900
400 y*8 2+
y8
b6/y5 DGPCGTVLTR

as their intensity. Additionally, peptide y3 b4

m/z
a y
200 y
4 y*6 7
and protein assignments obtained from 700 y
a2 1 b 2 y8
3 y*
database searches were included in the 0
7

maps. In total, 780 and 660 proteins 500 C Albumin

were identified in the adult head and the ETYGEMADCCAK

Intensity
embryo, respectively. A comparison 300 30 2+
y5
y y
2+ 7
a4 y 10 6

between the two maps showed that 307 a b 2 a y

y8 y9
100 2 3
4
of these were in common between the 0
two samples. Total numbers of proteins 2 3 4 5 6 7 8 9 0 200 400 600 800 1000 1200
m/z
for each sample as well as their overlap Drift time (ms)
as a function of gene ontology (GO) cel-
lular component are shown in FIGURE 6. Figure 4. 2D tD(m/z) plot (left) of tryptic peptides obtained from human urinary proteins. The plot
Differences in the numbers of proteins was obtained for components with a retention time of 23.75 min. Three MS/MS spectra shown on the
right are obtained by integrating all bins over narrow drift time ranges centered at (A) 3.73, (B) 4.38 and
for specific cellular components have (C) 5.09 ms. The data were obtained over the 23.25–24.00 min retention time range. These MS/MS spectra
been discussed. For example, it was sug- correspond with fragmentation of doubly charged peptide ions from the proteins xanthine
gested that the observance of an dehydrogenase/oxidase, uromodulin and albumin, respectively. Reproduced with permission from [22],
increased number of mitochondrial pro- © 2003 American Chemical Society.
teins in the adult head is corroborated MS/MS: Tandem mass spectrometry; m/z: Mass-to-charge ratio.
by findings from studies suggesting
mitochondrial densities are higher in neurons than other with 5-h LC runs). Due to the scanning nature of the MS
types of cells [42]. Other explanations for such observations instrument, multiple runs were required to obtain missed
were also discussed. assignments. A partial analysis of the 2D-LC-IMS-MS
approach described here allowed the identification of approxi-
Human plasma experiments mately 400 proteins from a 3 h 20 min experiment. Clearly,
With the advances in LC-IMS-MS methods mentioned above, the approach holds great promise with regard to experimental
an ambitious undertaking to characterize the human plasma throughput, which is increasingly important for profiling
proteome using LC-IMS-MS methods is currently underway. efforts. That is, many experiments may be required to address
Initial experiments have utilized a 2D-LC approach to analyze normal protein variabilities.
a whole-plasma digest (i.e., abundant proteins were not Another advantage of the 2D-LC-IMS-MS approach for
removed). In all, ten SCX fractions were analyzed using a proteomics analysis (particularly useful for plasma) is that
short, 21-min LC gradient. Overall, 2–7 × 104 features were features associated with low-abundance species can be
observed for each LC run and 598 unique peptides corre- observed in the presence of coeluting higher abundance spe-
sponding to 399 proteins have been identified from a partial cies as they are often separated by the mobility dispersion.
analysis of the data [36]. This leads to an effective increase in the experimental
It is instructive to compare such results with those obtained dynamic range. FIGURE 7A shows a 2D tD(m/z) plot obtained
for plasma using state-of-the-art LC/MS/MS instrumenta- over a 1 min tR range for the analysis of a single SCX plasma
tion. In head-to-head comparisons, the number of resolved fraction. To demonstrate the utility of the mobility disper-
and identified features from LC-IMS-MS experiments was sion, a mass spectrum obtained by integrating all bins for a
observed to be more than ten- and twofold higher, respec- narrow drift time range is shown in FIGURE 7B. Here, a feature
tively. The 2D-LC-IMS-MS analysis of plasma may also be that is buried in the noise in the total mass spectrum
compared with the work reported by Smith and coworkers, (obtained by integrating all bins over the entire drift range) is
who recently used LC/MS/MS and 2D-LC/MS/MS tech- clearly resolved. The MS/MS data obtained for this feature
niques to identify over 1600 proteins in a plasma sample – a provided an identification for the peptide MLVNFILR2+
significant achievement [43]. For their experiments, a single from the protein interleukin-26 (FIGURE 7C). Such assignments
SCX-LC/MS/MS analysis yielded approximately 600 protein are indicative of the high sensitivity of the LC-IMS-MS tech-
identifications requiring a 75-h experiment (15 SCX fractions nique, as cytokines have nominal plasma concentrations in the

www.future-drugs.com 557
Valentine, Liu, Plasencia et al.
pg/ml range. Thus, it is possible to obtain assignments for pep-
tides from proteins of vastly different plasma concentrations (a
108–109 concentration range). Capabilities such as these should
significantly enhance plasma profiling efforts by allowing the
monitoring of very low-abundance species.
Although the results for plasma analysis are quite promising,
a note of caution is necessary: probability-based scoring algo-
rithms such as that used by MASCOT for the present studies
are prone to contain false-positive assignments. Thus, it
becomes necessary to visually inspect the MS/MS data for ques-
tionable assignments. Also noted is that the ability to resolve
low-abundance peptides such as the one described above
requires high on-column sensitivities (attomole range). Previ-
ously, attomole detection limits have been reported for ion
mobility studies using direct infusion of peptide mixtures [21].
Since these early experiments, ion signals have been improved
by over one order of magnitude. Thus, it is likely that the LC-
IMS-MS method has the requisite sensitivity to observe very low-
abundance species. In related studies on human plasma, other
low-abundance proteins have been observed, including those
with multiple tryptic peptide assignments [UNPUBLISHED DATA].
The data from the 2D-LC-IMS-MS analysis of plasma have
also been compiled into an initial proteome map. The map
contains the position delineators and intensities for all observed
features as well as the peptide and protein information for iden-
tified species. The creation of the map is the first step towards
the use of LC-IMS-MS data for comparative proteomics and
individual profiling studies. The current status and future
developments envisioned for LC-IMS-MS mapping techniques
are described below.
Drosophila melanogaster embryo (0–2 h)
9.9 9.9
8.7 8.7
Drift time (ms)
Drift time (ms)
7.5 7.5
6.3 6.3
5.1 5.1
3.9
2.7
0 35 70 105 140
Retention time (min)
Figure 5. 2D tR(tD) datasets for a LC-IMS-MS analysis of a Drosophila melanogaster embryo
digest. The data were taken at the 0–2 h time point of embryogenesis. (A) was obtained by integrating
the total ion signal for each tR and tD value. (B) is a 2D base peak plot obtained by plotting the most
intense features for each tR and tD value.
IMS: Ion mobility spectrometry; LC: Liquid chromatography; MS: Mass spectrometry.
558
Current limitations
IMS-MS peak capacity and resolution
Although the 2D peak capacity of LC-IMS is quite high (see
above), IMS and MS measurements are correlated, leading to a
smaller IMS contribution (∼five- to tenfold for the current
instrument) to overall 3D peak capacity. While an order of
magnitude improvement with no significant sacrifice to signal
is quite good, peak capacity advantages associated with IMS
are more apparent for the parallel collision-induced dissocia-
tion (CID) method (i.e., the fragmentation of mobility-dis-
persed precursors). Here, the correlation is broken as the paral-
lel fragmentation allows the use of the entire drift range (peak
capacities of ∼25 for the experiments described above).
Improving the peak capacity further will require improvements
in IMS resolution (discussed below).
One of the problems associated with peptide identification
results from the relatively low resolution of the current mobility
separation. Since many features are not completely resolved in
the drift dimension, MS/MS spectra may contain undesired,
overlapping fragment peaks. Such features may hinder peptide
assignments, especially where there is a significant difference in
precursor abundance. This problem can be addressed using two
different approaches. First, more of the information from the
LC separation can be utilized to better correlate fragments with
precursors. Such information may include peptide retention
time persistence. That is, only fragments that persist in tR as long
as the precursor will be used in a MS/MS query. Currently, only
a match to retention and drift time is required. Similarly, peak
intensities may also be used; MS/MS data sets for given precur-
sors will not contain fragments that are in disproportionate
abundance. While such methods utilize
data processing algorithms, instrumental
approaches also exist. For example, the
resolution of the instrument can be
increased by incorporating a longer drift
tube. Recently, the resolving power has
been increased by factors of 2–3 without
significant loss in ion transmission through
the drift region [UNPUBLISHED DATA]. This is
accomplished using an ion funnel at the
back of the drift tube similar to the method
reported by Smith and coworkers [36].
3.9
2.7 Informatics
0 35 70 105 140 Currently, the rate of data generation from a
Retention time (min)
single LC-IMS-MS proteomics experiment
is quite large (0.2–1 Gbyte/min). Thus, for
a 21-min LC separation, a 21 Gbyte raw
data file can be generated. Such a generation
rate not only requires a large capacity for
data storage but also sophisticated algo-
rithms for data processing. Successful imple-
mentation of the instrumental improve-
ments described above will exacerbate the
computing hardware and software needs.
Expert Rev. Proteomics 2(4), (2005)
 Developing LC-ion mobility-MS techniques

120

111
100 76
59
48
95
80 61 24 34
68
Number of proteins

65
35
31

60
38
14
28 29 4
40 23
30 18 26
15 15 19 2
21 11 10
9 11 7 10
7 10
8 8 1
20 5 5 4 2
2 5 3 4 1
1 3 0 2
1 2 2
1 1 0 0

0
Cell cortex

Cytoplasm

Cytoskeleton

Cytosol

Endoplasmic reticulum

Extracellular

Extrinsic to membranes

Golgi apparatus

Integral to membranes

Membrane

Miochondrion

Nucleus

Plasma membrane

Rhabomere

Ribosome

Symaptic junction

Other
Synaptic vesicle
GO cellular component

Figure 6. Bar graph of the number of proteins observed for each Drosophila melanogaster developmental stage as a function of GO cellular
component. Purple and orange bars correspond to the adult head and embryo samples, respectively. The blue bar represents the overlap between the two
samples. Reproduced with permission from [41], © 2005 American Chemical Society.
GO: Gene ontology.

Over the last 2 years, significant progress has been achieved [UNPUBLISHED DATA]. Although the reduction in analysis time is
with regard to data analysis and should be noted here. To appre- quite significant (several months’ work previously), it is noted
ciate such progress, it is necessary to describe the data analysis that improvements are being pursued. Such improvements
process from sample collection to data set feature identification. include parallelizing processing algorithms, increasing the
Currently, the overall process is being carried out on a 24-node, fidelity of peak picking algorithms, as well as optimizing pro-
dual central processing unit (CPU; Opteron, 2.4 GHz) compu- tein database searching on the cluster. A goal would be to reach
ter cluster. After the collection and transfer to the cluster of a the same level of throughput on the cluster for data generated
LC-IMS-MS data set, algorithms create 3D data arrays contain- simultaneously from multiple IMS-MS instruments.
ing features observed for parent ion or CID conditions. A second The enhancement in data analysis has allowed the allocation
set of algorithms finds the positions and intensities of features of more resources for another critical informatics component –
observed in the parent ion and CID data arrays. Next, fragments knowledge assembly. Aspects of this work include the con-
are linked to their precursors via their mobilities and MS/MS struction and curating of LC-IMS-MS proteome maps (see
data sets are created for protein database searches. Finally, results below) and the comparison of data sets from disease samples
from protein database searches are grouped with features in the against such maps to determine molecular markers indicative
3D parent ion array to create initial analytical maps. of physiologic state. The recent study of human plasma has
With the recent automation of the processes described resulted in the construction of an initial plasma proteome map
above, the data analysis bottleneck has been significantly (see below) [UNPUBLISHED DATA]. Preliminary informatics work
reduced. For a recent study of human plasma involving a SCX- involves the use of commercial software as well as code devel-
LC-IMS-MS approach, the time required for feature identifi- oped in-house to determine protein and peptide correlations
cation was roughly equivalent to the experimental run time across the multiple LC runs comprising the emerging map.

www.future-drugs.com 559
Valentine, Liu, Plasencia et al.

This work is also focusing on comparisons between data sets
from different individuals as well as comparisons of single indi-
viduals against the growing plasma map. Such developmental
work is beginning to lay the foundation for more elaborate
approaches aimed at biomarker discovery.
Future directions
Instrumentation development: increasing LC-IMS-MS ion
signals & IMS resolution
Although the addition of the ion funnel has dramatically
improved the overall ion signal, instrumentation improvements
are envisioned to increase the current signal by approximately
tenfold. Instrumentation improvements will focus on the ion
storage capacity as well as the ion transmission efficiency in the
first drift region. The trapping efficiency (currently ∼10%)
may be increased by incorporating the novel ‘hour-glass’ ion
funnel design described by Smith and coworkers [36]. Such a
device affords greater ion storage capacity than that utilized for
the studies described here. Increased ion transmission effi-
ciency will be achieved using field-focusing strategies, as
described previously [40,44–46].
Other instrumentation development efforts will improve the
resolving power of the mobility separation. The resolving
power is given approximately as:
1
t
---
LEze -⎞ 2
----D- = ⎛ -----------------------
Δt ⎝ 16k B T ln 2⎠
where E, z, e and kB correspond to the drift electric field, ion
charge state, elementary charge and Boltzmann’s constant, respec-
tively [47]. A number of groups have pioneered high-resolution
IMS measurements [16,48–52]. Initially, high-resolution IMS meas-
urements were achieved by using high pressures as such condi-
tions allowed the use of higher voltages without creating a dis-
charge in the buffer gas. Recently, relatively high-resolution
measurements for peptides have been reported using low-pressure
IMS instrumentation [36]. This is accomplished by substantially
lengthening the drift tube and using an electrodynamic ion fun-
nel to collapse the diffuse ion cloud near the exit aperture. There
are two distinct advantages for using a low-pressure, high-resolu-
tion drift tube. First, a low-pressure mobility region is easily cou-
pled to ion trapping devices necessary for high sensitivity meas-
urements [35–36]. The second advantage is that CID experiments

1400
A 3.7 ms B
Intensity (arbitary units)

MLVNFILR2+
1200 from IL-26

1000

800
400 450 500 550 600
m/z

m/z

600 y(5)
C
Intensity (arbitary units)

400 y(7)

y(6)
200 y(4)
y(2) y(3)

0
2 4 6 8 200 400 600 800 1000 1200
m/z
Drift time (ms)

Figure 7. (A) 2D tD(m/z) plot for data collected from a LC-IMS-MS analysis of a single SCX fraction of a human plasma digest. The data set was obtained at a
retention time of 20.4 min. (B) A parent ion mass spectrum obtained by integrating all drift bins for given m/z values over the drift range indicated by the dashed
line box is shown. The feature corresponding with the precursor ion that has been identified by protein database searches as the ion MLVNFILR2+ from the protein
interleukin-26 is indicated. (C) The MS/MS spectrum for the MLVNFILR2+ ion indicating fragment assignments is also shown.
IMS: Ion mobility spectrometry; LC: Liquid chromatography; MS: Mass spectrometry; MS/MS: Tandem mass spectrometry; m/z: Mass-to-charge ratio.

560 Expert Rev. Proteomics 2(4), (2005)


45
40 +7
Flight time (ms)
+8
35 +9
+10
30
25
20
100 0 0 2 4
Drift time (ms)
Figure 8. Nested tD(tF) distributions showing charge state and conformational distributions of cytochrome c ions that were stored for two different
trap times (29 [left] and 75 [right] ms). Summed mass spectra and summed drift time distributions for each ion trap time are shown to the left and below,
respectively, of each 2D plot. Adapted with permission from [60], © 1999 American Chemical Society.
can be performed within the mobility region, such as those
demonstrated with the split-field drift tube [39]. Recently, an
extended (∼1 m), low-pressure drift tube employing the split-field
design for parallel CID has been used to perform LC-IMS-MS
experiments for human plasma [UNPUBLISHED DATA].
Developing LC-IMS/IMS-MS technology
The advantages of the combination of a post-ionization, rapid
gas-phase separation with LC/MS methods have been described
above in terms of increased peak capacity, dynamic range and
sensitivity, as well as the ability to generate a high-resolution
proteome map. An extension to this approach is to couple mul-
tiple gas-phase separation strategies similar to achievements
with condensed-phase separations [53]. Use of a second dimen-
sion of mobility separation requires the alteration of an ion’s
mobility, which can only be accomplished by a structural transi-
tion, or a change in buffer gas composition or temperature.
Since mobility measurements are carried out at high pressures, it
is possible to induce highly controlled structural transforma-
tions for biomolecular ions. For example, studies have shown
that ion structures can be varied by changing the energy used to
inject into a drift tube [58–59], or by changing the buffer gas tem-
perature [60–62]. Recently, it has also been shown that structures
vary depending upon ion storage time in traps (prior to injec-
tion into a drift tube) [63,64]. FIGURE 8 shows structural transfor-
mations for several charge states of electrosprayed cytochrome c
ions stored in a 3D trap for long time periods [63].
Although various methods exist for altering ion mobilities
(above), several are preferred for IMS/IMS instrumentation
development due to their simplicity as well as their amenability
for coupling with existing instrumentation. For example, a sim-
ple approach would be to alter the structures of ions (similar to
transformations demonstrated in FIGURE 8) after a first mobility
separation step simply by injecting them with varying energies
www.future-drugs.com
Developing LC-ion mobility-MS techniques
45
40 +7
Flight time (ms)
Evidence for
+8
unfolding in
35 +9
+10 the trap
30
25
29 ms 75 ms
20
6 8 100 0 0 2 4 6 8
Drift time (ms)
into a second drift tube. Another approach can be developed
using separate but uninterrupted mobility regions such as that
demonstrated with the split-field drift tube design shown in
FIGURE 2. As discussed above, when the fields in the second
region of the drift tube are increased substantially, ions are colli-
sionally heated and undergo fragmentation. Interestingly, the
mobilities of ions can be influenced using a field regime
between that used to induce fragmentation and that used for
the low-field mobility separation. The nature of the mobility
change as ions leave the low-field mobility region is not fully
understood at this point; however, such field alterations may
provide another means for IMS/IMS separations.
FIGURE 9 shows a hypothetical mobility device based on a dou-
ble split-field drift tube design. The combination allows for sev-
eral experimental analyses. First, ions separated according to their
low-field mobilities in the first drift tube may be transmitted as
precursors or induced to undergo fragmentation at the back of
drift tube one. In the second drift tube, the precursors may be
subjected to a high-field mobility separation or the precursor
fragments from the first separation may be subjected to a low-
field mobility separation. At the back of the second drift tube,
the twice dispersed precursors or the fragments may be transmit-
ted or again induced to undergo CID. Thus, such experiments
may provide a vast array of information such as IMS(low-field)-
IMS(high-field)-MS/MS analysis of precursors as well as
MS/MS/MS information for mobility-dispersed fragments.
Proteome mapping
A goal of proteomics studies is to develop a proteome map, a
searchable database of component identifications that can be
used in comparative studies. As mentioned above, the multiply
dispersive approach allows the construction of a higher resolution
map than those afforded by traditional LC/MS methods. An
advantage of the multidimensional separations described here is
561
Valentine, Liu, Plasencia et al.
that the extra feature delineator (tD’s from IMS and IMS/IMS
measurements) can be used to aid identification efforts and
should facilitate comparisons across multiple data sets increasing
the accuracy of the map. Additionally, the higher throughput
afforded by the LC-IMS-MS measurements allows the genera-
tion of a greater number of data sets that, upon comparison,
should also increase the confidence of mapped features. That is,
higher peptide assignment confidence is garnered for identical
features observed multiple times; similarly, the observation of
multiple peptides from a single protein across various data sets
increases the confidence associated with the protein assignment.
Conclusions
Although relatively new, the use of multidimensional LC-IMS-
MS techniques to analyze complex mixtures is proceeding at a
remarkable pace. For example, the first multiply dispersive LC-
IMS-MS analysis of a complex mixture of peptides was carried
out in 2001 [19]. Over the next few years, the analysis was
extended to complex proteome mixtures [21,22,26,36,40,41]. Recent
comparisons with LC/MS approaches have shown advantages
with regard to resolution, dynamic range and experimental
throughput. Here, it is also noted that improvements (described
above) are envisioned to significantly increase the overall peak
capacity and sensitivity of these methods. Altogether, LC-IMS-
MS techniques are poised to move from the arena of fringe
technologies to that of mainstream proteomics tools.
Expert commentary & five-year view
Over the last few years, improvements in IMS-MS instrumen-
tation have made it possible to perform LC-IMS-MS proteom-
ics analyses. While only a relatively few numbers of studies have
been carried out to date, the advantages in experimental
throughput, resolution and dynamic range discussed in this
paper should prompt greater efforts in technique development.
It is conceivable that where there are only a few research groups
Field 1
Octopole
linear trap
Figure 9. Schematic diagram of a hypothetical mobility region for an IMS/IMS instrument. The mobility region comprises two split-field drift tubes40 for
efficient separation and fragmentation of ions.
IMS: Ion mobility spectrometry.
562
promoting and developing IMS technology for complex bio-
logic mixture analysis today [19–26,36,45,65–68], the number may
increase by more than one order of magnitude over the next
5 years. It is also conceivable that commercial instrumentation
utilizing IMS in a dispersive fashion may be marketed during
this time frame. Such an event would likely lead to more rapid
growth in the numbers of groups using LC-IMS-MS techniques
for proteomics studies.
Although the robustness of LC-IMS-MS instrumentation has
been demonstrated by many of the experiments described
above, the most pressing issues limiting its use are related to the
present state of informatics tools. Innovative data processing
and mapping algorithms are currently under development and
it is now possible to go from data collection to proteome map
generation within a single day. Present resources allow the
simultaneous analysis of multiple data sets on the time scale
required for data collection. Although such efforts are beginning
to bear fruit, development will continue throughout a 5-year
time frame. A final aspect of the informatics tools required for
these studies is the ability to determine statistically significant
differences across multiple data sets. Although to date little has
been accomplished involving comparison algorithms for IMS-
MS data, initial development has begun and will accelerate over
the next 5 years. With these informatics tools in place, results
from multiply dispersive studies will be ready for a knowledge
assembly informatics piece, similar to current proteomics
efforts. Here, work will focus on extracting correlations that
illuminate molecular signatures and roles associated with disease
onset and progression.
Acknowledgements
The authors acknowledge partial funding of this work from the
National Institutes of Health (1R01GM-59145-03 and
GM59148), the National Science Foundation (CHE-0078737)
and the Indiana Genomics initiative (INGEN).
Drift tube 1 Drift tube 2
Field 2 Field 1 Field 2
First conical Second conical Differential
lens assembly lens assembly pumping
Expert Rev. Proteomics 2(4), (2005)
 Developing LC-ion mobility-MS techniques

Key issues

• Multidimensional liquid chromatography (LC) ion mobility spectrometry (IMS) mass spectrometry (MS) technology provides
advantages in resolution, throughput and dynamic range for the study of complex mixtures.
• Proteome mapping studies are facilitated by LC-IMS-MS techniques as higher resolution, high-confidence maps can be generated.
• Limitations in LC-IMS-MS technology related with feature identification can be reduced with improvements in IMS resolving
power as well as improvements in software. Informatics limitations require more computing power and algorithm development for
parallel processing.
• Future work may focus on the development of instrumentation that will execute 2D-IMS/IMS separations. Such strategies, coupled
with condensed-phase separation steps, should provide unprecedented resolution of complex mixture components and lead to the
development of high-confidence maps.

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