The intelligent protein informatics platform
Thermo Scientific Proteome Discoverer software

Transform proteomics mass
spectrometry data into insights
Thermo Scientific™ Proteome Discoverer™ software enables comprehensive
proteomics data processing workflows empowered by artificial intelligence.

• Powerful and flexible framework: Optimized analysis for data generated by the sophisticated acquisition methodsof Thermo Scientific™ Orbitrap™ mass spectrometers. Easily analyze MS2 and MSn data or multiple fragmentation
modes in the same analysis. Dig deeper into data using artificial intelligence with INFERYS™ Rescoring and theCHIMERYS™ intelligent search algorithm. Mix and match multiple search engines in series or in parallel to maximize
insights with built-for-purpose workflows. Use third party-developed nodes within an extensible framework.
• Study management: Integrated management of raw data files, search results, and quantification methods. Map

study factors to quantification channels to set up replicates and statistical analyses to easily match results toexperimental variables.
• Biological annotation: Match identified proteins with gene ontology, gene symbol, and pathway terms. Further

enhance biological interpretation using enrichment analysis to help transform data into biological insights.
• Powerful and flexible result visualization and interpretation: Results are presented hierarchically, with dynamic

links between proteins, peptides, peptide spectrum matches (PSMs), and quantification tables. Easily filter data
to view only those results of primary interest. Use interactive data plots that directly link to their source tables todynamically interact with results. Find which proteins or peptides are up- or down-regulated through the volcano
plot, which samples are related via a PCA plot, or visualize abundance patterns across files using the heat map.

2

Since releasing in 2009, Proteome Discoverer software has been featured in thousandsof publications across multiple proteomics applications including:• Bottom-up peptide and protein

Identification• Label-Free Quantification (LFQ)• Isobaric Mass Tagging (including

Thermo Scientific™ TMT™ and
Thermo Scientific™ TMTpro™) Peptide
Quantification• Single-cell proteomics• Immunopeptidomics• Metaproteomics

Proteome Discoverer software in peer reviewed articles

18000

15917

16000

• Top-down Proteoform Identification

and Quantification with Thermo
Scientific™ ProSightPD™ software

14000• Crosslinked peptide identification

10000

using XlinkX software• Post-Translational Modification(PTM) Analysis

13006

12000

10561
8428

8000

6611

6000
4000
2000
0

9
2009

79 261 637
2010

2011

2012

2194
1300
2013

3433

2014

4886

2015

2016

2017

2018

2019

2020

2021

3

Transform proteomics mass spectrometry
data into insights
Integrated study management
Performing complex proteomics experiments requires theability to map data files to experimental study conditions
to organize and make sense of results. Proteome
Discoverer software uses a built-in study design feature
to assign qualitative and quantitative variables. The study
allows selection of data files, association of the data files
with a quantification method, the creation of experimental
variables as study factors, and the mapping of study
factors to data files and quantification channels. Eachdata file or collection of fractionated data files can bemapped to one or more study factors and then used toperform comparative and statistical analyses.
Proteome Discoverer software enables the creation and mapping of sample types, study factors,
and quantification channels to data files for simplified and integrated study management.

Pierce™ TMT11plex yeast
digest standard

Parental
control

Knockout stains

4 strains ofsaccharomyces
cervices
met6Δ

his4Δ

ura2Δ

BY4741

11 TMT channels
technical replicates
N=3 or 2 (for BY4741)

Samples combined into
1 LC-MS analysis

126

MS1

127N

Isolation window

MS3

127C
128N

Quantify

128C
129N
129C
130N
130C
131
131C

m/z

Schematic representation of Pierce TMT11plex Yeast Digest Standard and replicates as
an example of study management with Proteome Discoverer software. The standard iscomposed of four Saccharomyces cerevisiae strains: three lines respectively lacking thenon-essential proteins Met6, His4, or Ura2, and the parental strain BY4741 for reference.1

4

Generate ratios from study variables or sample types for comparativeand statistical analyses.

Pre-defined and customizable
processing workflows

Powerful data processing workflows tailored to fit advanced instrument
acquisition strategies

The Proteome Discoverer software workflow
system is composed of a node-basedarchitecture that can connect in linear or
branched pathways to maximize the amountof information that can be gleaned from aproteomics dataset. The workflow systemallows for new users to quickly and easily
deploy pre-defined workflows for TMT, LFQ,
PTM identification, and crosslinked peptide
identification and offers flexibility for expert
users to assemble advanced workflows toanalyze the most complex proteomics data
sets. Spectra are introduced into the top of
the workflow and pass through nodes that
perform filtering, peptide identification,
quantification, false discovery rate (FDR)
calculations, PTM site localization,bioinformatic analysis, and more.

Proteomics experiments can require multiple fragmentation types to generate thenecessary information for a given study. Proteome Discoverer software supports all
Orbitrap mass spectrometer fragmentation methods for MS2 or MS n data including
higher energy collisional dissociation (HCD), collision-induced dissociation (CID),
electron transfer dissociation (ETD), electron transfer higher energy collisional
dissociation (EThcD), ultraviolet photodissociation (UVPD), proton transfer charge
reduction (PTCR) and combinations thereof. Uncover more peptides and proteins using
the Thermo Scientific™ FAIMS Pro Duo interface for identification and quantitation.
Customize workflows for multiple MS n modes with alternating CID and ETD
fragmentation using the Scan Event Filter and multiple search engines to improveannotation. Deploy the most confident TMT quantification strategies with synchronous
precursor selection MS3 (SPS MS3) and Real-Time Search-Aided TMT SPS MS3 with
Sequest™ HT and INFERYS Rescoring, the CHIMERYS intelligent search algorithm, orComet and use the Reporter Ions Quantifier node to quantify. Use MS2 and MS3
information to identify and quantify crosslinked peptides with the XlinkX node. Perform
top-down proteomics with the best sequence coverage with a combination of UVPD,
ETD, EThcD, HCD, CID, and PTCR with ProSightPD software. Identify the most PTMs
with multi-stage activation and EThcD using Sequest HT and IMP-ptmRS.

The workflow system in Proteome
Discoverer allows for customizable
workflows to analyze complex proteomics
data. The workflow system is composedof nodes that can connect in linear or
branched pathways. While theseworkflows are highly customizable,
pre-defined workflows for common
workflows such as TMT, LFQ, PTM
identification and cross-linked peptide
ID are included.

Workflow

The consensus workflow adds additionalgrouping and validation steps along withbiological interpretation. The Protein
Annotation node adds result tables togroup proteins with pathway annotation
and GO and Pfam annotations for built inbioinformatic analysis, such as enrichment.
Workflows can also be customized using
the Scripting Node to run executables tointegrate results from custom scripts
directly into Proteome Discoverer
software results.

Pre-built Proteome Discoverer
software workflows

Real-time
Search Aided
TMT SPS MS 3

PWF_Tribrid_TMT_Quan_SPS_MS3_
SequestHT_Percolator,
PWF_Tribrid_TMTpro_Quan_SPS_MS3_
SequestHT_Percolator

Can also use Comet search engine tomore closely match the RTS results

LFQ

PWF_Hybrid_LFQ_INFERYS_Rescoring_
SequestHT Percolator_2stage, PWF_
Tribrid_Precursor_Quan_and_LFQ_IT
HCD_SequestHT_Percolator,
PWF_Hybrid_LFQ_CHIMERYS

Also supports FAIMS LFQ

PTMs

PWF_Hybrid_SequestHT_MSAmanda_
Percolator_ptmRS.pdProcessingWF

Identify and localize PTMs andcan be used with quan nodes

Immunopeptidomics

PWF_Hybrid_INFERYS_Rescoring_
SequestHT_Percolator

INFERYS Rescoring helps confirm
peptides that have low Xcorr but highcorrelation to the prediction spectrum
for the identified peptide

MS2-MS 3 analysis DSSO_MS2_MS3_TMT6quanof cleavablecrosslinking

Spectra are streamed from the top of theworkflow and pass through nodes that
perform filtering, peptide ID, quantification,
FDR calculation, PTM site localization,bioinformatic analysis, and more.

Additional benefit

Top down
analysis

PSPD 1 percent FDR LFQ for Med HI data,
PSPD Truncation Subsequense Searchwith FDR, PSPD Single Proteoform

Proteome Discoverer software canquantify crosslinked peptide datausing either TMT or LFQ
Enables identification andquantification of large (>50 kDa)
proteoforms. Also supports PTCR.

Utilize pre-built Proteome Discoverer software workflows to easily turn data intomeaningful results.

5Comprehensive search capabilities
empowered by artificial intelligence
A deeper mining of proteomics data
by reimagining the analysis of tandem
mass spectra

Select up to 5 PSMs from sameMS2 spectrum

Deconvolution of chimeric spectra withartificial intelligence

Annotated MS2 spectrum

Combined predicted spectrum

Improvements in protein and peptide identification using artificial intelligence

Deconvolution and visualization of chimeric spectra. The experimental and predicted spectrum can be visualized by
selecting up to 5 PSMs at a time to display a mirror plot.

Continuous innovation to dig deeper into data
Peptide groups+61%141378

+11%
+125%

29586

74008

+9%

+91%

33505

30655

+20%
25542

3802

3474

+9%

5893

+41%

+9%

PSMs

66611

Proteins

54045

CHIMERYS intelligent searchalgorithm
workflow
Proteome
Discoverer
3.0 software

4168

The new CHIMERYS intelligent search algorithm utilizes
artificial intelligence to rethink the analysis of tandem
mass spectra from the ground up. CHIMERYS overcomesthe challenges of mixed peptide MS2 spectra by providing
accurate peptide spectrum and retention time predictions
through deep learning combined with a powerfuldeconvolution algorithm. This revolutionary approachleads to a deeper mining of data and substantially
increases the number of PSMs found in data-dependentacquisition data. In comparison to previous strategies,
CHIMERYS finds more PSMs per tandem mass spectrum
and markedly improves the identification rate, with fewer
spectra returning no PSMs and many spectra returning
three or more PSMs. This results in a substantial
improvement in the number of unique peptides andproteins identified and quantified in typical proteomics
datasets compared to other approaches.

2.3

6

New CHIMERYS intelligent
search algorithm

2.4

2.5

3.0

2.3

2.4

2.5

3.0

2.3

2.4

2.5

3.0

TheCHIMERYSCHIMERYS
intelligent searchalgorithm
workflow produces70%
more proteins,produces
CHIMERYS
produces
CHIMERYS
produces
70%
more
proteins
more thanmore 5× as many
almost5x asas
many
more
than
2× moreunique peptides,
and2xalmost
PSMs
Proteomethan Proteome
peptidesPSMs
as Proteome
Discoverer
2.3 Discoverer
software usingunique
the same
datathanfrom a typical
90-minuterun.
software 2.3
Proteome Discoverer
Discoverer software 2.3
software 2.3

Not just software—
wide window
acquisition
Optimize instrument acquisition
methods with Proteome Discoverer
software with CHIMERYS

The data-dependent acquisition of proteomics samples traditionally utilizes narrow isolation
windows to reduce the co-isolation of multiple peptides due to limitations in the ability of searchengines to deconvolute chimeric spectra. In contrast to these traditional constraints, theCHIMERYS intelligent search algorithm utilizes artificial intelligence to rethink the analysis of tandem
mass spectra from the ground up to enable the deconvolution of complex chimeric spectra andenables new strategies of data acquisition. Employing wider precursor isolation windows for MS2
data acquisition paired with data processing using CHIMERYS allows for even larger increases in
performance for both Orbitrap/Orbitrap and Orbitrap/Ion Trap acquisition methods. Acquiring MS2
data with the Orbitrap provides the biggest improvements with isolation widths between
2–4 Th and Ion Trap data acquisition plateaus around 1.5 Th. The pairing of optimized dataacquisition and processing strategies provides synergistic benefits by increasing instrument
utilization through the simultaneous fragmentation of more peptides per tandem mass
spectrum collected and the deconvolution of the resulting chimeric spectra to accuratelyinterpret the resulting data complexity with CHIMERYS.

7

Improving confidence in search results using INFERYS Rescoring
To improve the confidence in peptide identification for Sequest HT, the INFERYS Rescoring
deep learning-based model calculates all predicted spectra from all target and decoy
hits and calculates figures-of-merit for the correlation of predicted and measured spectra.
This extra information generated by INFERYS Rescoring is added into the Percolator
calculation for FDR thresholds. This results in an improvement in the number of uniquepeptides and proteins identified and quantified in typical proteomics datasets comparedto other approaches.

Accurate predictions of fragment ion intensities enable the calculation of intensity-based
scores, which increases the confidence in results. The additional confidence provided
by INFERYS Rescoring is particularly important for immunopeptidomics, where there isa huge search space and many very similar peptides with similar properties. For example,
in a HLA Class I data set from a patient derived melanoma cell line immunopeptidomics
study, INFERYS Rescoring provides a 58% increase in PSMs and 55% increase in
peptide identifications compared to Sequest HT alone. INFERYS Rescoring also increases
y1
the confidence in identifications when very low protein loads are used leading to low
131.08177
1.0
While the additional confidence provided by INFERYS Rescoring
increases
the175.11932
number129.10257
y4
229.11931b2
abundanceMS2 spectra,y 3such as for
single-cell504.24258
proteomics.
By adding
extra figures ofy5442.26736
226.11935
159.07678
375.19995
110.07150
of identified and quantified proteins for all proteomics applications,
it provides
specific
0.5
443.27081 487.22668
570.36041 603.31079
201.12390
y1
131.08177
116.07075
314.17239
b3
505.24594
604.31421
y2
169.13391
y6376.20258
merit,
peptideswhich
mayotherwisebemissed1.0
175.11932
486.23196 can be rescued to improve the coverage258.14523
115.08676
129.10257
304.16275
674.34711
424.25574
459.24725
y4
benefits for single-cell proteomics, metaproteomics, and immunopeptidomics.
By
229.11931b2
y3
y5
504.24258
442.26736
226.11935
0.0
andidentify
andquantify
more
proteins
andpeptidespersample.
This
results
in
15–20%
159.07678
375.19995 400
110.07150
100
200
300
500
600
700
0.5
443.27081 487.22668
570.36041 603.31079
201.12390
m/z
including a metric for the fragment ion intensities, INFERYS Rescoring
can identify
116.07075
314.17239
b3
505.24594
604.31421
y2
169.13391
y6376.20258
486.23196
more
HT alone.
258.14523peptide
115.08676
304.16275IDs versus Sequest
674.34711
424.25574
459.24725
targets that are hard to separate from decoys based on classicalscores.
0.020220106_OTOT_1ug_HeLa_120min_ISO4_021.raw #818 RT: 0.6980 min
FTMS, 382.7190@hcd30.00, z=+2, Mono m/z=381.19443 Da, MH+=761.38158 Da, Match Tol.=20 ppm

20220106_OTOT_1ug_HeLa_120min_ISO4_021.raw #818 RT: 0.6980 min
FTMS, 382.7190@hcd30.00, z=+2, Mono m/z=381.19443 Da, MH+=761.38158 Da, Match Tol.=20 ppm

Intensity [counts](106[counts])
Intensity(106)

Consensus Reference Spectrum (Inferys)

Consensus Reference Spectrum (Inferys)

20220106_OTOT_1ug_HeLa_120min_ISO4_021.raw #818 RT: 0.6980 min
FTMS, 382.7190@hcd30.00, z=+2, Mono m/z=381.19443 Da, MH+=761.38158 Da, Match Tol.=20 ppm

100

200

Spectrum
Selector
Precursor
Detector

Experimental spectrum

1
2

Intensity [counts] (106)Consensus Reference Spectrum (Inferys)

1.0

0.5

110.07150
116.07075

4

INFERYS
Rescoring5

SequestHT
Sequest HT
Scoring

0.5
1.0
0.0
0.5
-0.5
0.0

Intensity-based
Rescoring

1.0

FDR
Estimation

INFERYS Rescoring

Intensity [counts] (106)60.5

Decoy PSMs

258.14523

115.08676

b2

100

129.10257

110.07150
116.07075

y1
y1
175.11932

131.08177

y1
b2b2
159.07678

b3

200
226.11935

169.13391

201.12390

200

229.11931b3

b3
258.14523

314.17239
y2
304.16275

442.26736

600

y4504.24258

443.27081 487.22668

376.20258
424.25574

400

459.24725

505.24594

486.23196

570.36041

314.17239
y2
304.16275

y2

300
y2

442.26736

700

y5603.31079
604.31421

y6674.34711

y 3?
375.19995

y4
504.24258

505.24594
486.23196
424.25574
459.24725
y4504.24258
442.26736
443.27081 487.22668

376.20258
424.25574

459.24725

505.24594

486.23196

314.17239
y2
300
304.16275

400

m/z

442.26736

400

424.25574
m/z

459.24725

y5603.31079
604.31421

570.36041

y5
603.31079
604.31421

y5

500

600

y4
y4
504.24258

443.27081 487.22668

376.20258

570.36041

y4

y3

yy33?
375.19995

600

443.27081 487.22668

376.20258

314.17239
y2
304.16275

500

m/z

y 3?
375.19995

0.0

486.23196
500

505.24594

570.36041

y5
y5
603.31079
604.31421

600

700

y6
674.34711

y6674.34711

y6

y6

y6
674.34711

700

700

Consensus Reference Spectrum (Inferys)

-0.5
1.0

y1
175.11932

131.08177

-1.0
0.5
1.0
0.0
0.5
-0.5
0.0

229.11931b2
y1
b2
226.11935 b3
159.07678
110.07150
201.12390
y1
131.08177
116.07075b3
169.13391
175.11932
258.14523
115.08676
129.10257
100
200
229.11931b2
226.11935
159.07678
110.07150
b3
201.12390
116.07075b3
169.13391
258.14523
115.08676b2
y1b3

Consensus Reference Spectrum (Inferys)

Percolator SVM scorey3375.19995

300

229.11931b2
226.11935
159.07678
110.07150
201.12390
y1
131.08177
116.07075b3
169.13391
175.11932
258.14523
115.08676
129.10257
229.11931b2
226.11935
159.07678
110.07150
201.12390
116.07075b3
169.13391

100
115.08676

Intensity [counts](106[counts])
Intensity(106)

Frequency

Target PSMsb3
258.14523

200

Consensus Reference
Spectrum (Inferys)
129.10257

FDR estimation

229.11931y1
175.11932

131.08177

-1.0
-0.5

-1.0

Percolator

169.13391

100

Consensus Reference Spectrum (Inferys)e.g. Spectral angle

226.11935
201.12390

115.08676

0.0Intensity [counts](106[counts])
Intensity
(106)

XCorr

Sequest HT

b2
159.07678

Consensus Reference
Spectrum (Inferys)
129.10257

Experimental
MS2 Spectrum

3

500

m/z

Consensus Reference Spectrum (Inferys)

1.0

Spectrum
Properties Filter

400

y1
175.11932

131.08177
129.10257

300

-1.0
-0.5
1.0
-1.0

b2
131.08177

100
129.10257

y1
y1
175.11932200b2
159.07678

226.11935

229.11931y2
314.17239
y2
304.16275
300
y2
314.17239
y2
304.16275

yy33
375.19995

442.26736

376.20258
y3
375.19995

y4
y4
504.24258

443.27081 487.22668

400

505.24594
486.23196
424.25574
459.24725
500
y4m/z442.26736
504.24258
443.27081 487.22668

376.20258
424.25574

459.24725

505.24594

486.23196

y5
y5603.31079
604.31421

600
570.36041

y5
603.31079
604.31421

y6674.34711

700

y6
y6
674.34711
y6

y4
y3

y6

y5

y3
y2

300

570.36041

y5

400

y3
375.19995

m/z

442.26736

500

yy44
504.24258

600

700

y5
603.31079
604.31421

8

Intensity [counts] (106)443.27081 487.22668
The INFERYS Rescoring workflow. Experimental MS spectra are scored by Sequest HT 201.12390
by matching
all theoreticalb and y ions, each weighted
equally by thesearch engine. The INFERYS
570.36041
314.17239
b3
505.24594
169.13391
y2
y6
376.20258
100
200
300
400
500
700
258.14523
115.08676
304.16275intensities, and correlates
424.25574
459.24725
674.34711
Rescoring node subsequently reads the peptide sequences identified
by Sequest HT, predicts
theirfragment
the 486.23196
predicted
spectrum to 600
the original. Values
m/z0.0
corresponding to this correlation are included as features in Percolator, helping increase confidence
whose fragment abundances closely match those from the prediction.
b3 for PSMs
y2
y6

2

0.5

110.07150
116.07075

b2

-0.5

y1

y5

y3
y4

-1.0

100

200

300

400

m/z

500

600

700

B

HLA Class I Peptides
SequestHT 1% FDR

Identifications

40000

SequestHT+ INFERYS Rescoring 1% FDR

36912

SequestHT+ INFERYS Rescoring 0.1% FDR

30864

30000

Target PSMs

6000

SequestHT 0.1% FDR

50000

Sequest HT with INFERYS Rescoring

Sequest HT

2000

20000

0

8257

10000
0

5430

3474

PSMs

8408
1974

Consensus Reference Spectrum (inferys_2.1_fragmentation)

Cy 4+
357.21399

1.0
Intensity [counts] (106)169.09750

0.5

b2+
143.08237

340.19846

226.11914

y 2+ 195.11356
189.12373 222.12361

0.0
y

b3+
240.13490
y 3+

b4+
297.15640

323.17194
311.20935

260.16122

+
2

b

+
3

y

+
3

394.20966b5+
368.19354

339.20285b+
4

437.25180 b6+
518.27087
465.24673
508.29013b7+
428.25119
483.25986
536.28174
y 5+

y

+

b5

-0.5

+
5

b 6+

y 7+
582.32544
579.32697

b8+
607.31226

564.31543

y 8+653.36316

724.41077

+

+

b8

b7

+

-1.0

y7

+

y4

200
AAPGAPAAV.(A2)

b

300
y

Mix and match search engines with anextensible framework
The extensible framework of Proteome Discoverer
software allows for flexible workflows and the deploymentof multiple search engines. Sequest HT is the default
search engine and is used to identify peptides in tandem
mass spectra. Sequest HT evaluates protein sequencesfrom the provided database and calculates a list of
peptides that could be present given the precursor
information. Sequest HT calculates a theoretical tandem
mass spectrum for this list of candidate peptides andcompares the theoretical spectrum to the experimental
spectrum to yield the Xcorr score for ranking. In addition
to Sequest HT, a series of third party search engines areavailable to use in Proteome Discoverer software.

400

m/z

500

600

700

5000
0

Percolator SVM score

4091

Peptide groups

AG20201030_SR_HLA_A0201_2IPs_02.raw #13436 RT: 23.3793 min
FTMS, 724.4005@hcd30.00, z=+1, Mono m/z=724.40045 Da, MH+=724.40045 Da, Match Tol.=0.02 Da

Decoy PSMs

10000

4000

Frequency

58640

60000

Frequency

A

Percolator SVM score(A) INFERYS Rescoring predicts peptides on-the-fly during
a workflow only for those peptides identified by Sequest
HT in the target and decoy database searches to provide
additional figures-of-merit to increase the confidence for any
given PSM. INFERYS Rescoring substantially improves theanalysis of HLA peptides due to the very large search
space required. At the same 1% FDR threshold, with theinclusion of INFERYS Rescoring, the number of PSMs
increase by 58%, and the unique peptides by 55%.
(B) INFERYS Rescoring predictions are done accurately
based on fragmentation method, including HCD and CID,
and collision energy. The prediction has also been
improved to include TMT and TMTpro. This results in an
increased Percolator SVM score for well matched Target
PSMs. (C) The mirror plot shows how close the predicted
spectrum below matches to the experimental MS/MS
spectrum above for the selected PSM.

Peptides identified by
CHIMERYS Intelligent
Search Algorithm

35429

52536

Peptides identified
by MSPepSearch
7352
PTMs,
semitryptic
peptidesThe flexible workflows of Proteome Discoverer software
allows for search engines to be used independently, inseries, or in parallel to significantly increase the numberof peptide and protein identifications.

The extensible framework of Proteome Discoverer software
workflows allows for the use of multiple search engines
to glean more results from your raw data. Tailor searchstrategies to increase peptides even further by using library
search or other search engines specializing in PTMs, suchas MSPepSearch.

9

Label-free quantification workflows
Support for single ormultidimensional separations

Predefined LFQ workflows

Peptide identification• “Add Files” or “Add Fractions”• Support for all Thermo Scientific• Multiple search engines

Comprehensive quantification capabilities

Orbitrap mass spectrometers• For multidimensionaldata, acquisition
RT• Precursor Detector
Integrate
intelligent data
strategies with sophisticated dataalignment happens for the same
• FAIMS Pro Duo Interface support• INFERYS Rescoring
analysis to maximize biological insights

fraction• CHIMERYS Intelligent Search

600
Intensity [counts] (103)

Key features/benefits of label-free quantification workflows

Algorithm

y 1+
175.11943b3+
286.13980

400
200

y2+
246.15616

b4+
399.22635

y 8²+

b4+
y 3+

y 8²+

0
-200

+
y 1+ b2 y +2-400

y 13²+
b10+
y 13+
y 5+
y 11²+ 741.35370 y 14²+
1062.52332
y 9+1481.70068
b11+
605.28857 636.28778
797.89709
1161.58557
1069.49976
y 6+
1482.69604
1464.67786
y7+
719.33319
y 10+
y 11+
y 8+
818.40234
y 12+1156.53687
y 14+
955.46283
1271.56799
1384.64392
1594.77869
y 12²+b5y3+
375.19730

y4

y5+ y11²+

b5+
y4+

y10²+

y 15²++

y12²+ y6

y14²+

b10+ y 9+

y 8+

1000
m/z
b

y

Unique approach for ratio
and p-value calculation

LFQ visualization tools

Statistical analysis andvisualization tools• Protein ratios calculated as• Chromatograms, aligned and• Volcano plots• “Background-based” p-value uses

distribution of background ratios
at different abundance levels to
calculate p-value

10

y 14+

y 12+

y 13+

500

unaligned

y 11+

y 13²+ADVLPLDSNHVNTEEAR.(D21)the median of peptide ratios
including replicates

y 10+

y 7+

b3+

-600

• PCA

• Abundance plots• Heat maps

• Extracted ion chromatograms• Sequence map

1500

Proteome Discoverer software enables LFQ analyses
with comprehensive capabilities. Both single ormultidimensional separations can be utilized through theaddition of files or fractions. Predefined LFQ workflows
provide support for all Orbitrap mass spectrometers aswell as support for data generated with the FAIMS Pro
Duo interface. The Minora Feature Detector node detects
and quantifies the isotopic clusters in the MS1 data andthen maps the clusters to PSMs from the search engines
used in the workflow across all data files. Proteome
Discoverer software also uses a unique approach forratio and p-value calculations by using a pairwise ratio
method to produce accurate ratios and a p-value
calculation that uses the distribution of “background”
proteins to determine whether or not any single protein isdifferentially expressed. To aid in the visualization andfiltering of LFQ results the software also includes
visualization and statistical analysis tools, such as
abundance plots and heat maps, with direct connectivity
to the results for dynamic filtering and plotting.

B

3000
Quantified proteins

LFQ workflows can utilize the CHIMERYS intelligent
search algorithm or Sequest HT with INFERYS Rescoring
to identify and quantify more proteins. By digging deeper
into the data to identify more peptides, CHIMERYS
paired with the Minora Feature Detector quantifies
more peptides and proteins. Similarly, adding INFERYS
Rescoring into workflows using Sequest HT adds figuresof-merit from the comparison of the predicted spectrafrom INFERYS Rescoring for each target and decoy hit to
the experimental fragmentation spectrum that produced
those PSMs. Percolator uses this information to further
separate the distribution of true matches from random
ones to produce more identified and quantified peptides
and proteins.

A

C

3180
2572

2000

2311
1749

1000

0

CHIMERYS INFERYS Sequest HT
Rescoring Prec Det
CHIMERYS

40

SequestHT● Human● Yeast● Shared−4
INFERYS
Rescoring

40
Log2 ratio

Identify and quantify more proteins andpeptides with artificial intelligence−4
Sequest HT
PrecDet40−4Sequest HT

4
0
−4
4

6

8

Log10 peptide abundance

One hour data-dependent acquisition of 250 ng HeLa/125 ng Yeast lysate analyzed in triplicate on an
Thermo Scientific™ Orbitrap Exploris™ 480 mass spectrometer. (A) A typical LFQ workflow using Minora
Feature Detector and CHIMERYS Identification. (B) The CHIMERYS intelligent search algorithm increases
the number of quantified proteins compared to other strategies for typical LFQ analyses. (C) The addition of
the CHIMERYS intelligent search algorithm allows for increased identification and quantification, particularly
for lower abundance peptides, resulting in improved sensitivity and a lower limit of quantification.

11

Integrated statistical analysis and visualization tools
The statistical analysis and visualization tools built into Proteome Discoverer software
allow for rapid filtering, visualization, and interpretation of results. LFQ results include
chromatograms, Principal Component Analysis (PCA) plots, heat maps, quantification
channel charts, and more. Quantification channel charts can quickly display changes
for a given peptide or protein group across different experimental variables in tandemwith chromatogram traces to visualize the detected peaks across all conditions.

Complex datasets in applications with samples containing diverse protein expression,
such as single cell proteomics, can be quickly visualized using multivariate tools such as
PCA plots and hierarchical clustering heat maps. These plots are highly interactive,
making it easy to select and filter sets of peptides or proteins that differentiate groups.
For example, the heat map and PCA plots can be used to classify different cell types insingle cell proteomics data.2

LFQ results presentation

LFQ results can be visualized with chromatograms, principal component analysis plots, heat maps, quantitation channel charts, and more within theProteome Discoverer software framework, allowing for quick and easy interpretation of results.

12

Motor neuron (single neurons)

30

Interneuron (single neurons)
HeLa (0.5 ng – 2,3 cells each)

100Principal component analysis showing thedifferentiation of HeLa, motor neurons, andinterneurons from single cell experiments.

-10

-20

-10

0

10
PC 1 (52.2%)

20

30

40

Sample clustering

Cellularheterogeneity?

F9, InterNeuron

F8, InterNeuron

F7, InterNeuron

F12, MotorNeuron

F10, MotorNeuron

F6, HeLa

F4, HeLa

Samples cluster
by cell typeF5, HeLa

Protein clustering

F11, MotorNeuron

PC 2 (12.5%)

20

Heat map analysis showing the differentiation of HeLa, motor neurons, and interneurons from single cell experiments.

13

Isobaric Mass Tagging (TMT and TMTpro)
Labeled Peptide Quantification
Default TMT methods

Support for advanced
acquisition methods

Predefined workflows• TMT 2plex, 6plex, iodo TMT 6plex,• FAIMS• Support for ion trap, hybrid and tribrid

10plex, 11plex, and TMTpro

instruments

TurboTMT with sophisticated data
Integrate intelligent data acquisition• strategies
16plex and 18plex• MS2 and MS3-based quantification•
TMT SPS MS3
analysis to maximize biological insights• Customize to increase IDs

Unique approach for TMT protein
quantification

Powerful visualization andanalytical tools

Extensibility• S/N-based• PCA plots• Connect to external tools via the• Peptide filters for improved• Volcano plots

accuracy

Scripting Node• Integration with MSstatsTMT via• Heat maps

• Summed protein abundances

custom script• Enrichment analysis• Scale to pool/control channel forlarger datasets
P08727: Keratin, type I cytoskeletal 19 [OS=Homo sapiens]

160
140
120

Abundance [a.u.]

Key features/benefits of TMT quantification workflows• Correction factors

100

100

100

100

100

95

96

95

96
90

100

105
96

105

105

112
93

90

97

93

97
90

83

86

83

80

94

97

103

97

95

97

75

60
40

24

22

26

32
24

37

32

38

32

38

38

36

20
0
Brid ge

Control 10h

Control 24h

Control 2hControl 6hheavy standard
Quan Channels

14

light standard

Virus 10h

Virus 24h

Virus 2h

Virus 6h97

Integrate intelligent data acquisition strategies
with sophisticated data analysis to maximize
insights
TMT and TMTpro reagents allow for the multiplexing ofproteomics samples with robust and efficient derivatization
for better quantitative accuracy and precision. Proteome
Discoverer software offers default TMT methods to matchthe various reagents to provide the most accurate andprecise results. Proteome Discoverer software offers
predefined workflows to support all Orbitrap mass
spectrometers with the capability to do both MS2 andMS3-based quantification, along with the ability tocustomize workflows to increase the number of identified
and quantified peptides and proteins. TMT protein
quantification is performed using signal to noise summed
protein abundances from the peptide to protein levelvalues for improved confidence in quantification accuracy
and precision. Larger datasets with multiple runs aresupported using scaling with pools or control channels.
Results are easily visualized and filtered and can becustomized using the Scripting Node, for example withintegration of the MSstatsTMT R libraries.

MS2 peaks
selected for MS3

MS3 quan spectrum

Visualization of TMT and TMTpro results
Multiplexing samples across different groups can provide extensive information but
can require the use of multivariate visualization to identify similarities and differences.
For example, the comparison of eight different human cell lines with and without torin
stimulation yields clustering by similarity that can be visualized with the built in PCA andHeat Map plots. Tying the study factors and samples together via study management
paired with the built-in visualization provides clear differentiation between groups, with
HCT116 and RKO as well as HeLa and Panc1 clustering together due to the similarityof the cell lines.

TMT accuracy and precision is enhanced by utilizing TMT SPS MS3 methods and thenumber of identified peptides is improved with Real-Time Search-Aided methods.
Proteome Discoverer software results allow for the visualization of MS2 fragment ions thatare selected for MS 3 and the visualization of the MS 3 quantitation spectrum for maximum
confidence in results.

Match Real-Time Search-Aided TMT SPS MS3 results with the
Comet search engine
The Real-Time Search-Aided TMT SPS MS3 functionality uses the Comet search engine
for intelligent triggering of MS3 scans based on the identification of a PSM in thepreceding MS2 scan to improve accuracy, increase the number of quantified proteins,and provide interference free spectra. The Comet search engine has been added into
the Proteome Discoverer software framework to more closely match the peptides
identified during acquisition and can be used to process data alone or in conjunction
with other search engines.

MCF7

HCT116 and RKO

Panc1
RPE1

HeLa

293T

U2OS

Improved identification and confidence for TMT workflows
Adding artificial intelligence capabilities to TMT workflows allows for additional
identifications and more confident quantification. Using the CHIMERYS intelligent
search algorithm in MS2-based TMT workflows will provide confidence that peptides used
for quantification are not derived from chimeric spectra, which would result in errors inquantification. Both CHIMERYS and INFERYS Rescoring can also help improve thenumber of identified and quantified peptides and proteins by digging deeper into the data.

The multivariate analysis of different groups, such as multiple cancer cells lines as seenin this PCA plot, is integrated directly into the results visualization. Three replicates foreight different human cell lines each with and without torin stimulation that are quantified
with TMTpro reagent labeling leads to clustering by cell lines that are similar and a slightshift in response to torin stimulation.3

15

Data visualization
Proteome Discoverer software offers an intuitive
interface for results interpretation, including exceptional
connectivity between data tables and plots to allow rapid
interpretation, filtering, and visualization for a faster time
to results.

Peptide views
Results are presented using a nested table where PSMs
are directly linked to unique peptides, proteins, andannotated MS n spectra in an interactive fashion.
This allows for dynamic filtering and connectivity acrosstables, graphical charts, spectra, and chromatogramsfor more thorough investigation and interpretation.

Post translational modification identification and visualization
Understanding the function of proteins requires the identification of not only unmodified peptide sequences but also
the localization and presence of PTMs. The Proteome Discoverer software framework includes the IMP-ptmRS searchnode, which uses the peptide sequence determined by the main search engine, calculates the masses of residues
considering all possible positional isoforms, identifies and detects the diagnostic fragment ions, scores the hits, andcalculates a probability of localization for each PTM.3 In addition, Proteome Discoverer software includes a chemical
modifications table and modifications dialog to easily
annotate additional peaks in tandem mass spectra forneutral losses and/or diagnostic ions. This uniquecapability streamlines the analysis and interpretation for
PTMs of interest. Further insights can be gleaned byusing an error tolerant search through the Mascot node.
The linked Modification Sites, Peptide Isoforms, andPSMs tables can be used to find differentially expressed
PTMs in complex proteomics data.
The protein sequence
coverage view shows thelocation of identified peptides
as well as identified andlocalized post-translational
modifications. If the Protein
Annotation node is used in
the Consensus workflow, thePTMs annotated in UniProt
for the selected protein will
be displayed.

The hierarchical nesting of tables helps organize results tolink proteins, peptides, and PSMs together to simplify thenavigation through complex results.

16

PTMs can be easily identified and visualized through the modification sites table, whichallows for the selection of peptide sequences and the display of peptide isoforms. Withineach peptide isoform the modification pattern can be identified and annotated through theuse of ptmRS site probabilities and best site probabilities score for site specificity andincreased confidence in annotation.

60
60

40

Integrated statistical analysis tools
Interpreting the results of large proteomics experiments
requires the ability to map study factors and samples torelevant statistical tests and ratio generation with flexible
filtering and visualization capabilities to determine therelevant experimental results. Proteome Discoverer software
allows for a hierarchy of study factor variables to handle
biological factors, experimental categorical factors, andtechnical replicates. In order to provide the best quantitativeand qualitative results, normalization and scaling can be
performed along with protein ratio calculations based oneither protein abundance or pairwise ratios. Statisticalanalyses can be performed with a t-test. Results can also
be visualized using volcano plots to show pairwise
comparisons of proteins or peptides with adjustable P-value
and log2 fold change values to select and filter based ondesired changes.

20
20

0
0

Activation type: CID(Collision
InducedActivation
type:
CID
Dissocation)(Collision
Induced
Dissocation) 2
2

Count
Count

40

Count
Count

Proteome Discoverer software offers multiple plotting
capabilities related to both raw data and data processing
results. Information about raw data can be used to optimize
sample preparation or acquisition strategies and can also
be used to troubleshoot. Distribution charts can display any
metric with scatter plots, histograms, and bar charts. For
example, the number of missed cleavages can be displayed
in a bar chart to confirm full protein digestion or a histogram
chart of Xcorr values can be used to show which PSMs
pass the FDR threshold filter, or a comparison of identified
peptides using different fragmentation techniques can beseen with a Venn diagram. These retrospective analyses
help offer a more complete understanding of the generationand processing of data to move forward with informed
decision making to deploy optimized sample preparation,acquisition, and processing strategies to achieve the best
data quality and results

35000
35000
30000
30000
25000
25000
20000
20000
15000
15000
10000
10000
5000
5000
0
0

0
1
2
0 groups 1- #missed2cleavages
PeptidePeptide groups - #missed cleavages

15

Activation type:
HCD (High
Activation
type:Energy
Collision
HCD(High Energy
Dissocation)
Collision
Dissocation)

15

46
46

3
3

1
1

2
2

0.500
1.000
1.500
0.500
2.000
1.000
2.500
1.500
3.000
2.000
3.500
2.500
4.000
3.000
4.500
3.500
5.000
4.000
5.500
4.500
6.000
5.000
6.500
5.500
7.000
6.000
7.500
6.500
8.000
7.000
8.500
7.500
9.000
8.000
9.500
8.500
10.0009.000
9.500
10.000Deeper insights into data

PSMs - XCorr
PSMs - XCorr

2
2 Activation type:
ETD (Electron
Activation
type: Transfer
ETDDissocation)(Electron Transfer
Dissocation)

Distribution charts can be utilized to visualize, interpret, and optimize experimental procedures, data acquisition
settings, and results. For example, a bar graph can display the number of missed cleavages, a histogram can show theXcorr values for PSMs passing the pre-set FDR filter (dark red) and excluded hits (salmon), and Venn diagrams candisplay peptide sequences identified by different fragmentation techniques.

Confidence interval chart with abundance ratios

Volcano plot

Interactive abundance ratios comparing groups can be visualized with shading according to the confidence interval,
allowing for a more confident interpretation of proteome changes across a large dynamic ratio range using a
background-based p-value calculation. Volcano plots allow for the visualization and selection of proteins or peptides
in comparisons with customizable p-value and log2 fold change values.

17Annotation and pathway mapping
In addition to the identification and quantification of peptides and proteins, Proteome
Discoverer software provides biological interpretation of results. The addition of theProtein Annotation node to the consensus workflow creates the Pathway Protein Groups
and Annotation Protein Groups result tables. In addition, proteins can be selected from
Volcano Plots, PCA plots, and Heat Maps to generate an Enrichment table to display
overrepresented or underrepresented pathways, GO, and Pfam terms to quickly convert
results into biological insights. The annotation service is regularly updated to provide themost up to date terms. The service also allows full proteome FASTA file downloads andallows for easy updates over time as entries are modified in the database.

GO Annotation: identify biological processes, cellular components,and molecular functions. Map results onto pathways to understandbiological context.

Interactive enrichment table from graphical results to find overrepresented
pathways
Check all up-regulated
proteins in volcano plot

Enrichment table automatically updates
based on checked protein list

Up-regulated
Reactome pathways
Interactive enrichment tables allow for the selection of up- or down-regulated proteins or
peptides in any view, including volcano plots, PCA plots, and heat maps, and thevisualization of corresponding Reactome pathways from the checked proteins to help
derive biological interpretation of results. In addition to Reactome pathways, KEGG and
Wikipathways as well as gene ontology terms and protein families can be visualized in theenrichment table.

18

An annotation service with frequent updates allows for the display of overrepresented
pathways, GO, and Pfam terms associated with selected proteins.

Pathways can also be directly visualized, providing biological context and background
literature references using pathway maps.

Protein crosslinking with XlinkX

Key features for XlinkX forProteome Discoverer software

Combine crosslinked peptide identification with TMT and LFQ analysis for quantitative structural biology insights
Crosslink modifications

User interface forcrosslink modification
definition

Predefined workflows

LFQ and TMT
quantification

Annotated XlinkX
spectra

Export to third party
tools• Predefined crosslinks• Users can add• Cleavable and non-

• Uses same nodes as• Chromatograms, aligned• xiView

their own cross-linker• Cleavable and non-cleavablecleavable• Support for different

for standard proteomics
workflows

acquisition modes• Heterobifunctional

and unaligned• PyMOL• Abundance plots• Extracted ion

crosslinkers

chromatograms

Protein crosslinking enables structural proteomics studies to be performed using mass spectrometry to pinpoint residues in close proximity to the interaction interfaces between
protein complexes and interfaces. The XlinkX nodes in Proteome Discoverer software allow for easy analysis of crosslinked peptide data, supporting both cleavable and noncleavable crosslinkers. These nodes can also be combined with the TMT and the LFQ nodes in Proteome Discoverer software for quantification. * XlinkX developed in collaboration with
Utrecht University.

Top-down proteomics with ProSightPD software
Key features of ProSightPD software

Comprehensive top-down data analysis and visualization, supporting multiple fragmentation modes, FAIMS Pro Duo interface, and detailed proteoform analysis
Prebuilt and Flexible
Top-down workflows

Supports advanced
acquisition methods andquantification

Powerful databasemanagement

Versatile proteoform
identification

Unique top-down results
visualization

Extensive reporting
capabilities• Discovery LC-MS• PTCR• ProSight Annotator• Fast ProSight database• Visualizations from• TD Viewer• Native top-down• LFQ• Complex-down MS,

4

and more• ETD, EThcD, UVPD
• FAIMS Pro Duo Interface– Precise control of all
proteoforms– Intuitive user interface

search algorithms– Searches include
truncated forms andunknown modifications
via Δm mode.
– Supports unresolved
precursors

Proteome Discoversoftware• ProForma Annotated

Sequences• TD Validator• Fragment Map View

ProSightPD software enables the identification, characterization, and quantification of Top-down proteomics datasets within the Proteome Discoverer software framework. ProSightPD
software includes a Database Manager to import proteins of interest and add or remove isoforms or PTMs in tandem with workflows equipped to address the challenges of finding andannotating known and unknown proteoforms, including processing capabilities for all fragmentation modes such as HCD, CID, ETD, EThcD, UVPD, and PTCR, along with FAIMS Pro Duointerface data. Mix and match search types and deconvolution strategies to match acquisition and processing strategies to identify intact proteoforms and sub-sequences ofproteoforms. Visualize and filter results with the capabilities of Proteome Discoverer software including automated discovery and characterization of proteoforms and instantly validate
results with TD Validator.

19

ALM

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Thermo Scientific Orbitrap mass spectrometers
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1

Schweppe et al, Anal. Chem. (2019), 91, 6, 4010–4016.

2

https://pubs.rsc.org/en/content/articlehtml/2021/sc/d0sc03636f

3

Li et al, Nature methods https://doi.org/10/1038/s41592-020-0781-4

4Taus, T. et al, J Proteome Res., 10, 5354-5362 (2011). doi: 10.1021/pr200611n

Thermo Scientific Vanquish Neo UHPLC system
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Learn more at thermofisher.com/proteomediscoverer
For Research Use Only. Not for use in diagnostic procedures. © 2022 Thermo Fisher Scientific Inc. All rights reserved. CHIMERYS and INFERYS
are trademarks of MSAID. Mascot is a trademark of Matrix Science. SEQUEST is a trademark of the University of Washington. ProSightPD is a trademarkof Proteinaceous, Inc. Tandem Mass Tags (TMT) is a registered trademark of Proteome Sciences. All other trademarks are the property of Thermo Fisher
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