diaPASEF: parallel accumulation–serial fragmentation combined with data-independent acquisition

TLDR: This work uses the correlation of molecular weight and ion mobility in a trapped ion mobility device to devise a scan mode that samples up to 100% of the peptide precursor ion current in m/z and mobility windows and thereby increase the specificity for precursor identification.

Abstract: Data-independent acquisition modes isolate and concurrently fragment populations of different precursors by cycling through segments of a predefined precursor m/z range. Although these selection windows collectively cover the entire m/z range, overall, only a few per cent of all incoming ions are isolated for mass analysis. Here, we make use of the correlation of molecular weight and ion mobility in a trapped ion mobility device (timsTOF Pro) to devise a scan mode that samples up to 100% of the peptide precursor ion current in m/z and mobility windows. We extend an established targeted data extraction workflow by inclusion of the ion mobility dimension for both signal extraction and scoring and thereby increase the specificity for precursor identification. Data acquired from whole proteome digests and mixed organism samples demonstrate deep proteome coverage and a high degree of reproducibility as well as quantitative accuracy, even from 10 ng sample amounts. diaPASEF makes use of the correlation between the ion mobility and the m/z of peptides to trap and release precursor ions in a TIMS-TOF mass spectrometer for an almost complete sampling of the precursor ion beam with data-independent acquisition.

Figures

Figure 1 | The diaPASEF acquisition method. a, Schematic ion path of the timsTOF Pro mass spectrometer. b, Correlation of peptide ion mobility and m/z in a tryptic digest of HeLa cell lysate. c, In diaPASEF, the quadrupole isolation window (grey) is dynamically positioned as a function of ion mobility (arrow). In a single TIMS scan, ions from the selected mass ranges are fragmented to record ion mobility-resolved MS2 spectra of all precursors. d, Implementation of diaPASEF precursor selection with a stepped quadrupole isolation scheme. e, Representative example of a single diaPASEF scan with the precursor selection scheme from d.

Figure 3 | Ion mobility-aware targeted data extraction. a, Steps in the Mobi-DIK workflow to extract fragment ion chromatograms from diaPASEF scans. First, our workflow de-multiplexes diaPASEF data into individual diaPASEF windows and then uses the assay library coordinates to perform targeted extraction in the 4-dimensional analysis space of ion mobility, retention time and m/z. For each peptide precursor, a full extracted ion chromatogram, as well as an accompanying ion mobilogram and a set of high resolution MS2 spectra are extracted from the data and used for scoring. The OpenSWATH algorithm is used to perform peak group scoring, FDR estimation and alignment using the ion mobility enhanced data. b, Example chromatograms extracted at with and without restriction in the ion mobility dimension. c, Narrow extraction in ion mobility space improves signal-to-noise (S/N) and removes interfering signals from co-eluting precursors in the same diaPASEF window. The mean S/N ratio increases 2.6- to 4-fold when comparing full ion mobility extraction to narrow extraction. Boxplots show the median (center line), 25th and 75th percentile (lower and upper box limits), the 1.5 x the interquartile range (whiskers) and outliers (points). d, Relative number of detected peptide precursors at 1% FDR as a function of the ion mobility extraction window.

Figure 5 | Label-free protein quantification benchmark. HeLa digest was spiked with approximately 45 ng (sample A) and 15 ng (sample B) yeast digest and analyzed in triplicate diaPASEF single runs each. Log-transformed ratios are plotted as a function of protein abundance for n = 6,777 human and n = 708 yeast proteins. Boxplots show the median (center line), 25th and 75th percentile (lower and upper box limits), the 1.5 x the interquartile range (whiskers) and outliers (points).

Figure 4 | HeLa proteome analysis with diaPASEF. aNumber of peptide precursor ions in triplicate injections of 200 ng HeLa digest. b, Correlation of precursor ion mobility in a single diaPASEF run and the assay library. c, Relative deviation of ion mobility values in a single diaPASEF run to the precursor ion mobility in the library. d, Number of proteins inferred from the peptide precursors in a. e, Distribution of estimated copy numbers of proteins contained in the assay library and detected with diaPASEF in triplicate single runs. f, Completeness of the protein quantification matrix in triplicate single runs as a function of decreasing protein abundance.

Figure 2 | Efficiency of different data acquisition methods. a, Extracted fragment ion chromatogram of the y6 ion of the doubly charged DLGEEHFK peptide precursor in an LC-MS analysis of bovine serum album digest acquired with typical DDA and DIA methods as well as a 100% duty cycle diaPASEF method. b, Detected ion current from multiply charged precursors in single run analyses of a HeLa digest acquired with DDA, DIA and two diaPASEF schemes. To extract the ion current after quadrupole isolation, no collision energy was applied and the ion current was summed for each TIMS scan. The plot shows the rolling average of 60 TIMS scans.

Figure 6 | High duty cycle diaPASEF analysis of low sample amounts. a, Summed fragment ion intensity and coefficient of variation of shared peptides in triplicate injections of 10 ng HeLa digest with different diaPASEF duty cycles. b, Total peptide intensity distribution for both methods. c, Quantified proteins in n of 3 replicate injections of 10, 50 and 100 ng HeLa digest.

Full text

diaPASEF: parallel accumulation–serial fragmentation combined with
data-independent acquisition
Meier, F., Brunner, A-D., Frank, M., Ha, A., Bludau, I., Voytik, E., Kaspar-Schoenefeld, S., Lubeck, M., Raether,
O., Bache, N., Aebersold, R., Collins, B. C., Röst, H. L., & Mann, M. (2020). diaPASEF: parallel
accumulation–serial fragmentation combined with data-independent acquisition.
Nature Methods
,
17
, 1229-
1236. https://doi.org/10.1038/s41592-020-00998-0
Published in:
Nature Methods
Document Version:
Peer reviewed version
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Download date:09. Aug. 2022

1
Parallel accumulation – serial fragmentation combined with data-
independent acquisition (diaPASEF): Bottom-up proteomics with
near optimal ion usage
Florian Meier
1
, Andreas-David Brunner
1
, Max Frank
2
, Annie Ha
2
, Isabell Bludau
1
, Eugenia
Voytik
1
, Stephanie Kaspar-Schoenefeld
3
, Markus Lubeck
3
, Oliver Raether
3
, Ruedi Aebersold
4,5
,
Ben C. Collins
4,6*
, Hannes L. Röst
2*
and Matthias Mann
1,7*
1
Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany
2
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Canada
3
Bruker Daltonik GmbH, Bremen, Germany
4
Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
5
Faculty of Science, University of Zurich, Zurich, Switzerland
6
School of Biological Sciences, Queen's University of Belfast, UK
7
NNF Center for Protein Research, University of Copenhagen, Copenhagen, Denmark
*To whom correspondence may be addressed: ben.collins@qub.ac.uk, hannes.rost@utoronto.ca or
mmann@biochem.mpg.de

2
ABSTRACT
1
Data independent acquisition (DIA) modes isolate and concurrently fragment populations of
2
different precursors by cycling through segments of a predefined precursor m/z range.
3
Although these selection windows collectively cover the entire m/z range, overall only a few
4
percent of all incoming ions are sampled. Making use of the correlation of molecular weight
5
and ion mobility in a trapped ion mobility device (timsTOF Pro), we here devise a novel scan
6
mode that samples up to 100% of the peptide precursor ion current. We extend an
7
established targeted data extraction workflow by including the ion mobility dimension for
8
both signal extraction and scoring, thereby increasing the specificity for precursor
9
identification. Data acquired from whole proteome digests and mixed organism samples
10
demonstrate deep proteome coverage and a very high degree of reproducibility as well as
11
quantitative accuracy, even from 10 ng sample amounts.
12

3
INTRODUCTION
1
Mass spectrometry (MS)-based proteomics, like other omics technologies, aims for an unbiased,
2
comprehensive and quantitative description of the system under investigation
1–3
. Proteomics
3
workflows have become increasingly successful in characterizing complex proteomes in great
4
depth
4,5
. For the application of this technology to large sample cohorts e.g. for systematic screening
5
or clinical applications, which require a high degree of reproducibility and data completeness, data
6
independent acquisition (DIA) schemes are particularly attractive
6,7
. Unlike in data dependent
7
acquisition (DDA) where particular precursors are sequentially selected, in DIA groups of ions are
8
recursively isolated by the quadrupole and concurrently fragmented, thus generating convoluted
9
fragment ion spectra composed of fragments from many different precursors
8–10
. Although DIA
10
guarantees that each precursor in a predefined mass range is fragmented once per cycle, spectral
11
complexity poses a great challenge to subsequent analysis
11
. This is reduced by narrow isolation
12
windows, but these increase the cycle times needed to cover the entire mass range. Moreover, as
13
every precursor is only isolated once per cycle, the ion sampling efficiency at the mass selective
14
quadrupole for DIA methods is limited to 1-3% with typical schemes of 32 or 64 windows.
15
Adding ion mobility separation to the chromatographic and mass separation should increase
16
sensitivity and reduce spectral complexity
12–15
. The trapped ion mobility spectrometer (TIMS) is
17
a particularly compact mobility analyzer in which ions are captured in an RF ion tunnel by the
18
opposing forces of the gas flow from the source and the counteracting electric field
16–18
. Trapped
19
ions are then sequentially released as a function of their collisional cross section by lowering the
20
electric potential. Mobility resolution depends on the ramp time, which is typically 50 to 100 ms,
21
a time range between chromatographic peak widths (seconds) and the time-of-flight (TOF) spectral
22
acquisition (about 100 µs per pulse). In a TIMS-quadrupole-TOF configuration, the release of
23

4
precursor ions can be synchronized with the quadrupole selection in a method termed parallel
1
accumulation followed by serial fragmentation
19
. PASEF achieves a more than ten-fold increase
2
in sequencing speed in data dependent acquisition, without the loss of sensitivity that is otherwise
3
inherent to very fast fragmentation cycles
20,21
.
4
Here we investigate if the PASEF principle can be extended to DIA, combining the advantages of
5
this acquisition method with the inherent efficiency of PASEF. To realize this vision, we modified
6
the mass spectrometer to support ‘diaPASEF’ acquisition cycles. Building on open-source
7
software
22
we perform targeted extraction of fragment ion traces from the four dimensional data
8
space to confirm the identity and indicate the abundance of the peptides in the sample. We explore
9
the performance of the diaPASEF principle in typical proteomics applications such as single run
10
proteome analysis, label-free quantification, as well as the in-depth characterization of extremely
11
low sample amounts.
12
13
RESULTS
14
The diaPASEF principle
15
In the timsTOF Pro instrument (Bruker Daltonik), peptides separated by liquid chromatography
16
are ionized, introduced into the mass spectrometer and immediately trapped in a first TIMS device
17
(TIMS1, Fig. 1a). They are then transferred into TIMS2 from which they are released in reversed
18
order of their ion mobilities (largest ions released first). In parallel, incoming ions are again
19
accumulated in TIMS1, assuring full ion utilization. If operated in MS1 mode, the ion species
20
sequentially released from TIMS2 reach the orthogonal accelerator from which rapid TOF pulses
21
result in high-resolution mass spectra (>35,000 over the entire mass range). If operated in MS/MS
22