PASEF™ on a timsTOF Pro defines new performance
standards for shotgun proteomics with dramatic
improvements in MS/MS data acquisition rates
and sensitivity
Trapped ion mobility spectrometry coupled with quadrupole time-of-flight mass
spectrometry (timsTOF Pro) offers a unique dimension of characterization and
separation in complex mixtures. The timsTOF Pro instrument also enables the previously
introduced “Parallel Accumulation Serial Fragmentation” (PASEF) method.[1]
Keywords:
Trapped ion mobility
spectrometry (TIMS),
Parallel Accumulation
Serial Fragmentation
(PASEF)
Authors: Markus Lubeck1, Scarlet Beck1, Heiner Koch1, Stephanie Kaspar-Schoenefeld1, Niels Goedecke1, Oliver Raether1, Nicole Drechsler1, Michael Krause1,
Florian Meier2, Jürgen Cox2, Matthias Mann2.
1
Bruker Daltonik GmbH, Bremen, Germany;
2
Max Planck Institute of Biochemistry, Martinsried, Germany.
In the first publication on PASEF, a
proof of principle experiment was
performed using direct infusion,
which led to the prediction
that PASEF could achieve
dramatic improvements in
MS
/
MS data acquisition rates
in shotgun proteomics experi-
ments with improved sensitivity,
with the possibility to address
>170,000 precursors in a single
shotgun proteomics experiment.
This predicted performance far
exceeded the capabilities of
instruments available for shotgun
proteomics, providing results with
greater specificity and biological
relevance. In this application note,
we demonstrate that with further
hardware and software develop-
ments, the promise of PASEF has
now been realized, and that data
dependent shotgun proteomics
experiments performed using PASEF
can address 166,000 independent
precursors from a HeLa digest using
a 90 min gradient. The dual concen-
tration effects from the separations
by chromatography and trapped ion
mobility spectrometry provide a gain
of at least a factor of 10 in sensitivity
and using just 200 ng of HeLa digest
is sufficient to exceed what previously
required 1-2 µg of sample.[2] The
166,000 addressed precursors result
in more than 35,000 unique peptide
identifications and 5,500 protein identi-
fications from 200 ng of HeLa digest
injected on column. The benefits of this
approach for proteomics applications
are demonstrated using a timsTOF
Pro instrument (Bruker Daltonics)
with the PASEF acquisition mode
connected to a nanoElute UHPLC
(Bruker Daltonics).
Introduction
Multi-dimensional separations are
necessary to obtain a more complete
and accurate view of the content of
complex proteomics samples in both
discovery and targeted (quantitative)
workflows, and ion mobility has been
used as an additional separation
device in commercial mass spectro-
meters since the 1960s. More recently
trapped ion mobility spectrometry
(TIMS) has been introduced and
coupled to QTOF mass spectrometers.
In TIMS, ions are propelled through
a TIMS tunnel by a gas flow. An
electric field traps each ion at the
position where the push that it
experiences from the gas flow
matches the force of the electric
field, resulting in separation by mobility,
a function of their three-dimensional
size and charge in the gas phase
(Figure 1). Ramping down the electrical
field allows a selective release of
ions from the TIMS tunnel according
to their ion mobility (Ω/z), where Ω
is the ion’s collisional cross section
(CCS). In contrast to traditional drift
To mass analyzer, ions of highest mobility
(greatest collisional cross-section) eluting first
v
g
E
Parallel accumulation
TIMS scan
Figure 1: TIMS operation mode: With the
directed forces of gas flow, v
g, and electrical
field, E, ions are accumulated and trapped
according to mobility. Step-wise decrease
of the electrical field permits serial elution of
separated ions, which are then transferred
to the QTOF. Using the parallel accumulation
operation mode ions are eluted from the
second part of the TIMS device while the
next series of ions is introduced into the first
part of the TIMS device.
tube separation by size-to-charge, the
larger ions elute first, followed by ions
of decreasing cross section. In this
study, a timsTOF Pro instrument
(Bruker Daltonics) was used, with
the eluted ions further analyzed
via quadrupole time-of-flight mass
spectrometry (Figure 2). The spectra
generated during the TIMS elution
cycle is termed a TIMS scan. The
two dimensional separation of ions
(1/K
0 and m / z) summed across a TIMS
scan can be visualized as a TIMS MS
heat map, with mobility and m / z as the
y and x axes, respectively (Figure 3).
Likewise, particular ions may be
isolated in three dimensions according
to their liquid chromatography (LC)
retention time, TIMS elution time and
m / z (in this study, by a quadrupole
filter) and subjected to collision
induced dissociation. In the generated
PASEF MS / MS heat map, fragments
may be easily aligned with their
precursors in the TIMS MS heat map,
given that they appear at the same
ion mobility (Figure 3).
While this orthogonal separation can
provide a deeper look into complex pro-
teomics samples, its utility for peptide
identification may be challenged in
many instruments due to insufficient
sensitivity resulting from low ion
abundance (leading to weak spectra
from subsequent collision induced
fragmentation), as well as high scan-
ning speeds required to be compatible
with the typical 100 ms TIMS scan
times. Through the implementation
of the “Parallel Accumulation Serial
Fragmentation” (PASEF) method on
a fast scanning QTOF instrument,
fragment ion spectra from isolated
precursor ions may be acquired more
rapidly without sacrificing spectral
quality. By using a dual TIMS analyzer,
ions may be nearly continuously
queued for accumulation, sorting and
elution by mobility, allowing a duty
cycle near 100% (Figure 1). During
the timeframe of each TIMS scan, the
quadrupole switches its (mass) iso-
lation position several times, enabling
the collection of MS/MS spectra from
multiple precursors (Figure 4). Spectra
for low abundant precursors may be
summed to improve signal intensities
to increase both the number and
confidence of peptide identifications.
The use of ion mobility separation
prior to MS / MS results in improved
signal-to-noise ratios, as the targeted
ion species is time focused and
subsequently transferred to the
collision cell only during the periods of
time they elute from the TIMS device.
Additionally, by virtue of this additional
Second generation
dual TIMS analyzer
TIMS synchronized
quadrupole mass filter
UHR-TOF with high
efficiency technology
High speed
collision cell
Figure 2. Ion optics of the timsTOF Pro instrument including a dual TIMS analyzer and QTOF mass spectrometer.
dimension of separation, precursors
of the same m / z that co-elute from
the LC may be distinguishable which
is not possible with mass based
selection alone.
Two dimensional (m / z-mobility) data
dependent precursor acquisition
Another challenge in fast data
acquisition is the computational time
required to determine the precursors
that will be selected for MS/MS.
Maximizing the number of quality
MS/MS spectra, an essential step
in successful shotgun proteomics
experiments, requires consideration of
relative ion intensities and resolutions,
the latter of which is increased (thus
increasing the number of viable
precursors) with the second dimension
of separation. With higher speed data
acquisition capabilities, lower intensity
targets may be fragmented multiple
times. A rather simple and rapid
algorithmic approach[3] was developed
to efficiently schedule the subsequent
fragmentation scans based on
the initial detection of precursor
ions during the MS scan in both
the m / z and mobility dimensions.
Even in very complex proteomics
samples, computational time does not
exceed 1 ms, easily supporting high
throughput data collection.
Experimental
A complex tryptic peptide mixture
derived from HeLa cells was diluted
with 0.1% formic acid (FA) in water
to a concentration of 200 ng/µL. A
nanoElute UHPLC (Bruker Daltonics)
was coupled to the timsTOF Pro mass
spectrometer. The peptide mixture
(200 ng) was loaded onto a C18 column
(25 cm X 75 µm 1.6 µm, IonOpticks,
Australia). Chromatographic separation
was carried out using a linear gradient
of 2-37% of buffer B (0.1% FA in
ACN) at a flow rate of 400 nL/min
over 90 min. MS data was collected
over a m / z range of 100 to 1700, and
MS / MS range of 100 to 1700. During
MS / MS data collection, each TIMS
cycle was 1.1 s and included 1 MS + an
average of 10 PASEF MS / MS scans.
The acquired data were submitted
to the MASCOT search engine for
peptide identifications.
Rapid data collection...
As may be expected for this type of
sample, the base peak chromatogram
(BPC) (Figure 5, top) indicates an
extremely complex collection of pep-
tide species. Considering one narrow
time slice of the LC gradient (61.7 min),
the power of the PASEF method is
readily seen. During this example
scan, 41 different precursors were
fed into the scheduling algorithm and
addressed by a total of 10 PASEF
MS / MS scans. By examination of a
single TIMS elution (time) heat map
(Figure 5, bottom left), rapid quadru-
pole isolation window switching for
the first 12 precursors is evident.
Additional precursors are targeted in
subsequent segments of the elution
gradient. All precursors analyzed
within this example PASEF cycle are
indicated (Figure 5, bottom right),
including the resequencing of targets
of lower abundance. Within this single
PASEF cycle (1.1 s), the equivalent
of 119 separate MS
/
MS events
(if collected without PASEF on a
timsTOF mass spectrometer) were
conducted.
…while maintaining necessary
sensitivity
Important proteomic features are
often hidden within low intensity
peaks of complex samples, peaks
which might not be targeted by
instruments with slower MS
/
MS
acquisition rates. The sensitivity
(through the TIMS time focusing and
the ability to sum spectra of low
abundance targets) and separation
power (through differences in ion
mobility) of the PASEF approach is
Figure 3. Illustration of TIMS MS (left) and PASEF MS / MS (right) heat maps resulting from TIMS separations. Although the data generated may be very
complex, the characteristic ion mobility of targeted precursors and their fragments facilitates easy data alignment. Note that precursors of the same m / z
(yellow and green) may be clearly separated by their mobility.
1/K
0
[Vs/cm2]
1/K
0
[Vs/cm2]
m / z
m / z
PASEF MS/MS Heat Map
TIMS MS Heat Map
Figure 5. LC MS/MS run, 200 ng HeLa digest,
90 min gradient. A : Base peak chromatogram.
B : TIMS MS heat map at 61.7 min, with targeted
precursors indicated. Bottom left, first PASEF
MS/MS analysis event, precursors circled in
orange. Bottom right, full PASEF MS/MS cycle,
with each of the ten colored lines indicating
targets from one TIMS elution segment. The
most abundant precursors were sequenced
in previous scan cycles and were dynamically
excluded from re-sequencing.
m / z
Time (minutes)
10
40
70
30
60
90
20
50
80
100
1/K
0
400
1.4
0.8
0.9
1.0
1.1
1.2
1.3
1000
800
1400
600
1200
A
B
1/K
0
400
1.4
0.8
0.9
1.0
1.1
1.2
1.3
1000
800
1400
600
1200
Figure 4. Illustration of the PASEF method in
comparison with the standard TIMS MS / MS
operation mode, with the same 100 ms times-
cale. Using PASEF (lower panel), the quadrupole
switches its isolation position several times during
each ion mobility scan, with multiple precursors
isolated and subsequently transferred to the
collision cell. In contrast, with the standard
TIMS MS / MS approach (upper panel), only one
precursor from each TIMS scan is selected.
TIMS
T
IMS
M
S
/
M
S
P
ar
all
el
A
cc
u
m
u
la
ti
o
n
S
eri
al
F
ra
g
m
en
ta
tio
n
Quadrupole
CID
Spectrum Scan Time
100
ms
m / z
m / z
m / z
m / z
100
ms
m / z
TIMS tunnel
TIMS tunnel
TIMS tunnel
TIMS tunnel
TIMS tunnel
m / z
m / z
m / z
m / z
m / z
Figure 6. Collection of precursors at m / z
887.96, 888.94, and 889.78 indicated on a TIMS
MS heat map, with the number of summed
MS / MS spectra for each indicated. The base
peak chromatogram for this narrow m / z range
is shown in the bottom of the heat map.
Figure 7. Example of low intensity peptide
matches from HeLa cell analysis via PASEF
differ by only 2 Da. While their isotopic patterns
overlap in m / z, they are separated in the mobility
dimension and the PASEF MS / MS results yield
confident identifications.
1/K
0
M
ob
ili
ty
1.4
0.8
0.7
0.9
1.0
1.1
1.2
1.3
890
885
900
905 m / z
800
895
Precursor
Mass:
m / z 888.94 2+
CCS:
464.71 Å2
Intensity: 21219
Fragment
Repititions: 3
Mascot Ions Score: 62
Expectation Value: 6.7e-7
Precursor
Mass:
m / z 889.78 3+
CCS:
614.83 Å2
Intensity: 11844
Fragment
Repititions: 4
Mascot Ions Score: 72
Expectation Value: 6.9e-8
Precursor
Mass:
m / z 887.96 2+
CCS:
498.32 Å2
Intensity: 10355
Fragment
Repititions: 5
Mascot Ions Score: 35
Expectation Value: 3.0e-5
demonstrated in Figure 6. Summed
MS
/
MS spectra from three low
intensity precursors of similar m / z in
a dense region of the spectrum were
readily identified with good sequence
coverage, high MASCOT scores and
low expectation values (probabilities
to be a random match). Clearly, high
performance of the (subsequent)
QTOF is also critical, and the timsTOF
Pro mass spectrometer provides high
resolution, ppm accurate mass and high
isotopic fidelity (True Isotopic Pattern,
or TIP™). The benefit of this combined
approach for shotgun proteomics
applications is clearly indicated by
the increase in confident peptide
identifications (Figure 8). In the same
sample (200 ng HeLa digest, eluted
from the LC with a 90 min gradient),
nearly twice as many peptides were
identified using the PASEF approach
as compared to analysis without
trapped ion mobility spectrometry
separation (using the same instru-
mentation, however, with the TIMS
device switched off).
7A
7A
7B
7B
7C
7C
LC-Settings
LC-System
nanoElute UHPLC system (Bruker Daltonics)
Column
25 cm X 75 µm 1.6 µm C18 column
(IonOpticks, Australia)
LC flow rate
400 nL/min
LC elution conditions
2% to 37% (100% ACN, 0.1% FA)
Column oven (Sonation) temperature 50°C
Source
CaptiveSpray Ion Source (Bruker Daltonics)
Ionization
ESI (+)
Table 1 and 2: Instrumental Details
MS-Settings
MS-System
timsTOF Pro mass spectrometer (Bruker Daltonics)
TIMS elution
100 ms accumulation and ramp time (100% duty cycle)
Cycle time
1.1 s cycle with 1 MS + 10 PASEF MS / MS
Scan range
100 – 1700 m / z
Conclusions
“Parallel Accumulation Serial Fragmentation” (PASEF) on a timsTOF Pro
instrument (Bruker Daltonics) has been shown to successfully deliver
significantly higher speed and sensitivity in a data-dependent shotgun
proteomics workflow, as demonstrated by the analysis of 166,000
independent precursors from a HeLa digest using a 90 min gradient. By
applying PASEF, the number of identifications could be increased to more
than 40,000 unique peptide identifications and 5,500 protein identifications
from only 200 ng of HeLa digest injected on column. The number of
precursors able to be targeted with this approach is dramatically increased
by virtue of the rapid sample separation by both chromatography
and trapped ion mobility spectrometry. These exceptional acquisition
rates also support improved sensitivity, as low abundant precursors
may be targeted several times. This unique combination of features
provides a new standard for shotgun proteomics perfromance, enabling
researchers to find and identify more biologically relevant proteins.
Discussion
The recent introduction of trapped
ion mobility spectrometry (TIMS)
has added an innovative dimension
to quadrupole time-of-flight (QTOF).
The incorporation of the previously
described “Parallel Accumulation
Serial Fragmentation” (PASEF)[1]
method within Bruker’s proprietary
timsTOF Pro instrument significantly
magnifies these benefits. In this
experiment, more than 160,000
independent precursors from a HeLa
digest, separated using a 90 min LC
gradient, were addressed, with very
fast MS / MS data acquisition rates.
The timsTOF Pro used for this study
provides critical advances in separation
and sensitivity. Trapped ion mobility
spectrometry separation alone leads
to improved spectral quality by virtue
of the reduction of background noise.
The use of two regions of separation
within the TIMS device enables 100%
duty cycle so that no ions are lost, ions
are accumulated in the first region
of the TIMS device, while they are
scanned out into the QTOF from the
second region. The quadrupole of the
timsTOF Pro can switch its isolation
window on the millisecond time scale,
enabling the QTOF to address >10
precursors in a typical 100 msec
TIMS scan. The sensitivity is improved
through the additional time focusing
achieved by the TIMS, and additional
sensitivity for low abundance precur-
sors is achieved by addressing the
same precursor in multiple PASEF
scans.
Total
number
of MS/MS
spectra
Precursors
targeted
Peptide
spectrum
matches
Unique peptide
sequences, FDR
< 1%, (Mascot
with Percolator)
Protein
groups
(Mascot with
Percolator)
TIMS-PASEF 610,000
166,000
73,000
39,000
5,200
TIMS off
105,000
105,000
29,000
20,000
3,200
Figure 8. Protein ID results. Approximately six times as many MS / MS spectra can be collected in the
same time frame, with nearly double the number of unique peptide sequences and 1.5 times as many
protein groups represented.
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For research use only. Not for use in diagnostic procedures.
[email protected] – www.bruker.com
References
[1] Meier, et al. (2015) Parallel Accumulation - Serial Fragmentation (PASEF): Multiplying
Sequencing Speed and Sensitivity by Synchronized Scans in a Trapped Ion Mobility Device.
J Proteome Res. 14(12):5378-87
[2] Beck, et al (2015) The Impact II, a Very High-Resolution Quadrupole Time-of-Flight Instrument
(QTOF) for Deep Shotgun Proteomics. Mol Cell Proteomics. 14(7):2014-29.
[3] Lubeck et al. Evaluation of a peptide selection algorithm for trapped ion mobility with parallel
accumulation serial fragmentation (tims PASEF) on a QTOF instrument. Poster presented at:
65th ASMS Conference on Mass Spectrometry and Allied Topics, June 6, 2017; Indianapolis, IN.
Th
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P
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Bruker Daltonics GmbH & Co. KG
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