[Application Note]

Comparison of DIA and Shotgun

Quantitation on a Thermo Q Exactive
Roland M. Bruderer1,2, Oliver M. Bernhardt1, Tejas P. Gandhi1,
Saa M. Miladinovi1,2, Reto Ossola1, Tobias Ehrenberger1, Oliver Rinner1, Lukas Reiter1
1Biognosys, Zurich, Switzerland; 2 Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland
BiognoSYS AG | Switzerland | +41 44 738 2040 | www.biognosys.ch | info@biognosys.ch

Introduction
Until recently, quantitative proteomic discovery
mainly relied on data dependent acquisition (DDA)
also called shotgun proteomics. In DDA peptides are
selected for fragmentation at run time based on their
intensity. When utilizing DDA for experiments comprising
several conditions and replicates, many analytes will not be
consistently measured across all LC-MS runs. These missing
values pose a problem for precise quantitation. In contrast,
data-independent acquisition (DIA) yields complete and
reproducible maps. For DIA, the mass spectrometer cycles
through wide, pre-defined, contiguous precursor windows
and records high resolution fragment ion spectra thereof
[1,2]. With this method a large precursor m/z range can
be covered and a duty cycle of roughly 3-6 seconds can
be achieved in a highly reproducible manner. Unlike DDA,
the data is analyzed in a targeted mode [3]. Pre compiled

Methods
Sample Preparation. A 15cm dish of confluent human
embrionic kidney cell line (HEK-293) cells was lysed by
resuspension in 8M urea and 0.1 M ammonium bicarbonate
(to 1g/l protein). The lysate was reduced with 5mM TCEP
for 1 h at 37C. Subsequently, the lysate was alkylated with
25 mM iodoacetamide for 20 min at 21C. The lysate was
then digested with trypsin at a ratio 1:100 (enzyme:protein)
at 37C for 15h. Next the digestion was stopped with
trifluoroacetic acid. The samples were spun at 20000g
for 10 min at 4C. The peptides were desalted using C18
MacroSpin columns from The Nest Group according to
manufacturers instructions. After drying, the peptides
were resuspended in 1% acetonitrile and 0.1% formic acid
to 1g/l. The Biognosys HRM Calibration Kit was added to
the sample.
Liquid Chromatography. Peptides were separated on a
self-made 20 cm C18 analytical column (75 m x 15 cm)
packed with 3 m Bruker Magic C18AQ medium using an
Easy-nLC (Thermo Scientific). After equilibration with 3l 5%
acetonitrile 0.1% formic acid, the peptides were separated

spectrum libraries are used to probe the fragment ion maps.

Quantitation is performed on the fragment ion level (MS2) in
contrast to quantitation on precursor level in DDA (MS1). As
the DIA maps are always comprehensive, targeted analysis
will yield significantly less missing values for quantitation.
Further, quantitation on the fragment ion level has the
advantage that interferences are less problematic as it is
unlikely to obtain interferences for all fragment ions at the
same time for a single peptide. Targeted analysis of the
comprehensive DIA maps requires efficient algorithms and
software. To date, SpectronautTM is the most performant
software for the targeted analysis of such DIA data sets.
Spectronaut also provides powerful scoring, accurate peak
integration, reliable estimates of false discovery rates and
analysis across multiple runs [4]. We call DIA data analyzed
with Spectronaut Hyper Reaction Monitoring (HRM).

Window

Start

End

Window

Start

End

400

426

11

659

691

424

451

12

689

721

449

476

13

719

771

474

501

14

769

821

499

526

15

819

871

524

551

16

869

921

549

576

17

919

996

574

601

18

994

1071

599

631

19

1069

1146

10

629

661

20

1144

1220

Table 1. Precursor windows and corresponding start and end m/z

values used for DIA data acquisition in AIF mode on a Thermo Scientific
Q Exactive.

by a 2 hour gradient from 1% to 35% acetonitrile with 0.1%

formic acid at 300nl/min. After the gradient a linear increase
to 100% acetonitrile in 2 min was performed followed by 8
min 100% acetonitrile. The spectral acquisition duration was
130 min starting after equilibration.

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[Application Note]
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Data Acquisition. For DIA acquisition, a Thermo Scientific

Q Exactive was operated in sequential full scan and all ion
fragmentation window mode. The range of 400 to 1220 m/z
was covered by 20 sequential acquisition window pairs of
varying size (Table 1). The pairs consisted of a survey full
scan at 17500 resolution with AGC target of 1e6 ions (max.
injection time 1ms). All ion fragmentation was performed on
the same isolation window (AGC target 5e5 or 120ms max.
total injection time) at 35000 resolution. Fragments were
recorded from 200 to 1800 m/z. Stepped collision energy
was 10% at 25% normalized collision energy. Spectra were
recorded in profile type. The duty cycle for this DIA method
was 6.4s +/- 0.35s (n=3) which showed to be sufficient for
precise quantitation. For DDA a method from Olsen et al. with
minor changes was used. In brief, the Q Exactive instrument
was operated in data dependent mode to switch between
full MS and MS/MS acquisition. The full scan range was from
400 to 1200 m/z and the resolution at 70000 (m/z 200) after
accumulation 1e6 ion target (or 120ms max. injection time),
based on predictive AGC from the previous full scan. The top
12 most intense ions (8 z 2) per full scan were targeted
for fragmentation (2 m/z isolation window) in the octapole
collision cell by higher-energy collisional dissociation (target
5e5 ions or 60ms max injection time) at 17500 resolution
and from 200 to 1800 m/z. Stepped collision energy was
10% at 25% normalized collision energy. Dynamic exclusion

was set to 30s. The transfer capillary temperature was set

to 200 C with S-Lens RF at 60. Spectra were recorded in
profile type. A sequence of three HRM-MS and three DDA
runs (three technical replicates each) was shuffled prior to
acquisition in order to remove any biases of instrument or
LC performance.
Spectral Library Generation. The spectral library used
for HRM-MS was generated using 24 DDA runs. DDA data
was analyzed using a state of the art search engine and a
spectral library was generated using the Biognosys spectral
library generation pipeline Genesis (Table 2). Apart from
annotation of precursors and fragment ions the library
also contained accurate normalized retention times (iRT).
Spectral library generation is also available as a service for
academia from Biognosys.
Data Analysis. Shotgun data was searched using MaxQuant,
HRM-MS data was analyzed using SpectronautTM. False
discovery rate was set to 1% for both, DDA and HRM-MS.
Shotgun and HRM-MS responses were normalized prior to
analysis.

Total

Identified

Identified [%]

Proteins

4154

3576

86

Peptides

21369

17677

83

Precursors

24214

20246

84

Table 2. Total number of Precursors, Peptides and Proteins contained

in the spectral library used for HRM-MS. Cumulative unique
identifications of the spectral library in HRM-MS for the three technical
replicates (false discovery rate = 1%).

Results and Discussion

Figure 1. Human VCP in three technical replicates of HEK-293 cell line

protein extracts. HRM-MS data was analyzed with Spectronaut. (a) HRMMS raw data never contains gaps. (b) High reproducibility of relative
fragment ion intensitites across three technical replicates for the same
precursor as shown in (a).

In order to compare HRM-MS with DDA a HEK-293 cell line

sample was measured with three technical replicates using
HRM-MS or DDA on a Thermo Scientific Q Exactive. The
HRM-MS data was analyzed with SpectronautTM and the
DDA using MaxQuant. Both data sets were filtered with a
false discovery rate of 1% and responses were normalized.
Figure 1 shows HRM-MS raw data and automatically
picked peaks for a single precursor of the human valosin
containing protein (VCP) across three technical replicates.
Unlike DDA, where MS2 spectra can be missing in the
raw data, HRM-MS data contains no gaps. Therefore,
missing values occur mostly due to a signals dropping
below the limit of detection and not because a precursor
was not picked for fragmentation as for data dependent
acquisition.

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Figure 2 shows average number of identifications across

three replicate measurements for HRM-MS and DDA
respectively. Both data sets were filtered with a false
discovery rate of 1%. Number of Identifications was
significantly higher in HRM-MS as compared to DDA.
18173 vs. 16323 precursors and 3364 vs. 3245 proteins
were identified in HRM-MS and DDA, respectively. The
better performance of HRM-MS can be explained with the
detailed information available in the spectral library, such
as normalized retention time (iRT) and relative fragment ion
intensities that are typically not considered in DDA.
Figure 3 shows the higher reproducibility of HRM-MS by
comparing the number of full profiles across the three
technical replicates. A full profile is defined as a precursor

Identified Precursors and

Proteins
20000
18000
16000
14000
12000
10000
8000
6000
4000
2000
0

detected in all three replicates with a false discovery rate of

1%. HRM-MS and DDA yielded 16091 and 10806 full profiles
respectively, which corresponds to a 48% improvement of
HRM-MS as compared to DDA. The number of full profiles
has a large influence on reliable quantitation across
conditions and replicates. The precision of the measured
response and the profile completeness largely influence the
statistical power.
In Figure 4 we investigated the precision of the measured
response by means of coefficients of variation (CV). The
median CVs were 8.7% and 10.9% for HRM-MS and DDA,
respectively (p-value < 2.2e-16).

Median CVs [%]

Full Profiles
12

18000
16000

10

14000

12000
10000

8000
6000

4000

2000
Precursors
HRM-MS

Proteins
DDA

Figure 2. Average identified unique

precursors and proteins across the three
technical replicates for HRM-MS and DDA
respectively with a false discovery rate of 1%.
HRM-MS data shows more identifications
as compared to DDA due to the detailed
information available in the spectral library.

HRM-MS

DDA

Figure 3. Number of full profiles for HRMMS and DDA for three technical replicates
each. HRM-MS shows significantly more
full precursor profiles due to the high
reproducibility of DIA and the targeted data
analysis.

HRM-MS

DDA

Figure 4. Median coefficient of variation

(CV) for HRM-MS and DDA. CVs are
significantly lower for HRM-MS as compared
to DDA (p-value < 2.2e-16). Quantitation was
performed on MS2 and MS1 for HRM-MS and
DDA, respectively.

Conclusions
Here we showed that HRM-MS comprises the hallmarks for
a quantitative proteomics discovery technology: content,
reproducibility and precision was improved as compared to
DDA. HRM-MS will be especially useful in studies aiming at

quantitation across several conditions with a high statistical

power such as time course, drug screening, or dosage
experiments. This data suggests that HRM-MS is better
suited for label-free quantitative discovery as compared to
DDA.

References
1. John D. Venable et al. Automated approach for quantitative analysis of complex peptide mixtures from tandem mass
spectra Nature Methods 2004 Oct;1(1):39-45
2. Alexandre Panchaud et al. Precursor acquisition independent from ion count: how to dive deeper into the proteomics
ocean Anal. Chem. 2009 Aug;81(15):6481-8
3. Ludovic C. Gillet et al. Targeted Data Extraction of the MS/MS Spectra Generated by Data-independent Acquisition: A
New Concept for Consistent and Accurate Proteome Analysis Mol. Cell. Proteomics 2012 Jun;11(6):O111.016717
4. Oliver M. Bernhardt et al. Spectronaut: A fast and efficient algorithm for MRM-like processing of data independent
acquisition (SWATH-MS) data 2012; Proceedings 60th ASMS Conference on Mass Spectrometry
Discovery Proteomics | Precise Quantitation | DIA | Spectronaut | HRM Calibration Kit