such datasets.

The latest release in the Proteome Discoverer platform has several new features for
analysis of complex datasets. The first major feature is the new Consensus workflow
that creates persistent reports that open very large datasets quickly. Secondly, the
results are presented in a new hierarchical format with linked views for protein groups,
proteins, peptides, and peptide spectrum matches (PSMs). Third, the most critical
feature for analysis of large quantitative datasets is the new study management. These

Quantitative Analysis of Large

Poster Note 64491

will be demonstrated on a large dataset of a quantitative comparison between human
embryonic stem cell (hESC) and neural stem cell (hNSC) derivatives

Phosphopeptide DatasetsMethodsUsing
Proteins were extracted from hESCs and hNSCs, reduced and alkylated using

Proteome
Quantitative analysisDiscoverer 2.0
of large phosphopeptide datasets using Pro
iodoacetamide, digested using trypsin, and separated into 32 fractions using strong
cation exchange (SCX) chromatography. Phosphopeptides enrichment was
preformed, also collecting the flow through and wash fractions from the SCX fractions.
More details on the cells and the sample preparation will be available in a forthcoming F
a
David M. Horn, Ilyas Singec, Laurence Brill
David1 M. Horn,1 Ilyas Singec,2 Laurence
1
2 Brill2
T
Thermo Fisher Scientific, San Jose, CA, USA ; 2
2 Each of the fractions were analyzed in duplicate using a data dependent
publication.
decision tree LC/MS/MS method on an LTQ Orbitrap Velos mass spectrometer
Sanford-Burnham Research Institute, La Jolla, CA
a equipped with ETD.
1Thermo Fisher Scientific, San Jose, CA, USA, 2Sanford-Burnham Research Institute
n
Proteome Discoverer study design 1
a
The first step for data analysis in Proteome Discoverer 2.0 software is to create a new c
study and to list the study factors. For this study, there were 4 samples (3955 - hESC, (
3956 –hNSC, 3957 - hESC, and 3958-hNSC), two enrichment methods (TiO2 and t
IMAC), flow-through, wash and enriched fractions, each run twice leading to two
technical replicates. These were all entered as study factors as shown in Figure 1a. T
p
Overview The next
These studystep is towill
factors
new “Add Fractions”
quantification
choose
feature
values will
the raw
be used files. Proteome
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Discoverer
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2.0 software
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as a single sample. For
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Purpose: Interpretation of a complex quantitative phosphoproteomic dataset in the this study, each set of raw data files that fit the criteria of the study factors above such
a) b)
Proteome Discoverer 2.0 platform. as Sample 3955, TiO2-enriched phosphopeptides, technical replicate 1 are loaded
simultaneously as a single sample. A typical group has 25-50 data files, with the larger a
Methods: Phosphopeptides were enriched using IMAC or TiO2 from hESC and hNSC collection of data files corresponding to samples where the flow through and wash
samples and the flow through, wash and enriched fractions were collected. All MS samples from phoshpopeptide enrichment were run as separate LC/MS/MS runs.
data were acquired on a Thermo ScientificTM LTQ OrbitrapTM Mass Spectrometer Once the datasets were imported into the software, the study factors defined in Figure
Quantitative analysis of large phosphopeptide datasets using Prot
system equipped with ETD. Data were analyzed using the new study management
features in Thermo ScientificTM Proteome DiscovererTM software 2.0 using a label-free
1a were assigned to each sample as shown in Figure 1b above.
The third step is to create a new analysis, which includes two node-based workflows
quantification approach
David M. Horn, 1 Ilyas Brill Singec, 2 Laurence as 2well as the selection of the quantification details. Proteome Discoverer 2.0 software
Results: Proteome Discoverer 2.0 software processed the 4 stem cell samples in ~7 introduces a new dual workflow setup that includes a Processing Workflow for peptide
days with identification and quantification of phosphoproteins and phosphopeptides. identification, peptide quantification, FDR calculation and PTM site localization and
Thermo Fisher Scientific, San Jose, CA, USA, Sanford-Burnham Research Institute,
1 Several proteins and phosphopeptides are shown to be differentially expressed and
these are known to be regulated in stem cells as well as some novel proteins not
adds a new2 Consensus workflow that is used to perform protein inference, filter the
results based on FDR and other calculations, and summarize quantification results.
known to be differentially regulated. The two workflows used for this study are shown in Figures 2a-b below.
Processing Consensus F
Introduction FIGURE 1a) Study factors created for this analysis. b) List of samples imported
into Proteome Discoverer software and the study factors assigned to those f
With advances in liquid chromatography/mass spectrometry (LC/MS) technology, The r
samples. Each of these samples corresponds to at least 25 raw data files.
complexity of proteomics data is increasing rapidly. It is becoming increasingly
common to find datasets with 100’s of GB of raw data files for complex scientific
Overview
studies, putting an increased burden on downstream software tools for interpretation of
The next step is to choose the raw files. Proteome Discoverer 2.0 software includes a
new “Add Fractions” feature that groups a set of raw data files as a single sample. For
T
T
such datasets. this study, each set of raw data files that fit the criteria of the study factors above such
Purpose: Interpretation of a complex quantitative phosphoproteomic dataset in the f
Proteome Discoverer as Sample 3955, TiO2-enriched phosphopeptides, technical replicate 1 are loaded q
The latest release in2.0theplatform.
Proteome Discoverer platform has several new features for simultaneously as a single sample. A typical group has 25-50 data files, with the larger
analysis Phosphopeptides
Methods: of complex datasets. wereThe first major
enriched usingfeature
IMAC is orthe new
TiO2 Consensus
from hESC and workflow
hNSC collection of data files corresponding to samples where the flow through and wash F
that creates
samples and thepersistent reportswash
flow through, that and
openenriched
very large datasets
fractions quickly.
were Secondly,
collected. All MSthe samples from phoshpopeptide enrichment were run as separate LC/MS/MS runs. e
data were acquired on a Thermo Scientific LTQ Orbitrap Mass Spectrometergroups,
results are presented in a new hierarchical
TM format with linked
TM views for protein Once the datasets were imported into the software, the study factors defined in Figure f
proteins,
system peptides,
equipped withand
ETD.peptide spectrum
Data were matches
analyzed using(PSMs).
the new Third,
studythe most critical
management 1a were assigned to each sample as shown in Figure 1b above. s
featureinfor
features analysis
Thermo of largeTMquantitative
Scientific datasets isTMthe
Proteome Discoverer new study
software management.
2.0 using These
a label-free
The third step is to create a new analysis, which includes two node-based workflows
will be demonstrated
quantification approachon a large dataset of a quantitative comparison between human
embryonic stem cell (hESC) and neural stem cell (hNSC) derivatives as well as the selection of the quantification details. Proteome Discoverer 2.0 software R
Results: Proteome Discoverer 2.0 software processed the 4 stem cell samples in ~7 introduces a new dual workflow setup that includes a Processing Workflow for peptide P
days with identification and quantification of phosphoproteins and phosphopeptides.
Methods
Several proteins and phosphopeptides are shown to be differentially expressed and
identification, peptide quantification, FDR calculation and PTM site localization and
adds a new Consensus workflow that is used to perform protein inference, filter the T
these are known
Proteins to be regulated
were extracted in stemand
from hESCs cells as wellreduced
hNSCs, as some novel
and proteins
alkylated not
using results based on FDR and other calculations, and summarize quantification results. e
known to be differentially
iodoacetamide, digestedregulated.
using trypsin, and separated into 32 fractions using strong The two workflows used for this study are shown in Figures 2a-b below. A
cation exchange (SCX) chromatography. Phosphopeptides enrichment was e
Processing Consensus
Introduction
preformed, also collecting the flow through and wash fractions from the SCX fractions.
More details on the cells and the sample preparation will be available in a forthcoming FIGURE 2. Processing and consensus workflows for the phosphopeptide
F
r
With advancesEach
publication. in liquid chromatography/mass
of the fractions were analyzed spectrometry
in duplicate (LC/MS)
using atechnology, The
data dependent analysis.
complexity of proteomics
decision tree LC/MS/MSdata is increasing
method on an LTQ rapidly. It isVelos
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massincreasingly
spectrometer The processing workflow in Figure 2 includes Sequest HT nodes to interpret both CID
common
equippedto find
with datasets
ETD. with 100’s of GB of raw data files for complex scientific and ETD MS/MS and includes the Event Detector and Precursor Ions Area Detector
studies, putting an increased burden on downstream software tools for interpretation of nodes to calculate peak areas for identified peptides. All Sequest HT searches used
such datasets.
Proteome Discoverer study design 10 ppm precursor mass tolerance, while the CID data used 0.6 m/z fragment tolerance
and the ETD data used 1.2 m/z fragment tolerance. All searches used fixed
The
The latest
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step for in theanalysis
data Proteome DiscovererDiscoverer
in Proteome platform has 2.0several
softwarenew features
is to create for
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analysis
study andof complex
to list thedatasets. The first
study factors. Formajor feature
this study, is the
there new4 Consensus
were samples (3955 workflow
- hESC, (peptide N-term Q). Finally, the ptmRS node was appended to the end of the workflow
that creates persistent reports that open very large datasets
3956 –hNSC, 3957 - hESC, and 3958-hNSC), two enrichment methods (TiO2 and quickly. Secondly, the to calculate modification site localization probability.
results
IMAC), areflow-through,
presented inwash a newandhierarchical format with
enriched fractions, linked
each runviews
twice for protein
leading groups,
to two
proteins,
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spectrum matches
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as study Third,
factors as the most
shown incritical
Figure 1a. The Consensus workflow is used to calculate protein groups, filtering peptides and
feature
Thesefor analysis
study factorsof large
will bequantitative datasetsDiscoverer
used by Proteome is the new to study management.
determine which These proteins by false discovery rate, and rolling up the quantification results from individual
will be demonstrated
quantification valuesonwill
a large dataset of
be calculated a quantitative
and shown in the comparison
final report.between human peptide spectral matches (PSM’s) to peptide groups and proteins.
embryonic stem cell (hESC) and neural stem cell (hNSC) derivatives
a) b)
a) b)
Methods
Proteins were extracted from hESCs and hNSCs, reduced and alkylated using
F
iodoacetamide, digested using trypsin, and separated into 32 fractions using strong
c
cation exchange (SCX) chromatography. Phosphopeptides enrichment was
p
preformed, also collecting the flow through and wash fractions from the SCX fractions.
FIGURE 2. Processing and consensus workflows for the phosphopeptide p
More details on the cells and the sample preparation will be available in a forthcoming
analysis. p
publication. Each of the fractions were analyzed in duplicate using a data dependent
g
decision tree LC/MS/MS method on an LTQ Orbitrap Velos mass spectrometer The processing workflow in Figure 2 includes Sequest HT nodes to interpret both CID
equipped with ETD. and ETD MS/MS and includes the Event Detector and Precursor Ions Area Detector T
nodes to calculate peak areas for identified peptides. All Sequest HT searches used i
Proteome Discoverer study design 10 ppm precursor mass tolerance, while the CID data used 0.6 m/z fragment tolerance h
and the ETD data used 1.2 m/z fragment tolerance. All searches used fixed t
The first step for data analysis in Proteome Discoverer 2.0 software is to create a new carbamidomethylation, variable phosphorylation (S,T,Y), oxidation (M), and pyro-Glu h
FIGURE
study and to1a)
listStudy factors
the study created
factors. For for
this this analysis.
study, b) List
there were of samples
4 samples (3955 imported
- hESC, FIGURE 3. Study factors for the enriched (a) phosphopeptide search and the
(peptide N-term Q). Finally, the ptmRS node was appended to the end of the workflow i
The
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final report with the quantification values. This setup also works similarly for reporter for reporter ionion
asasSample
Sample 3955,
3955, TiO2-enriched
TiO2-enriched phosphopeptides,
phosphopeptides, technical
technical replicate
replicate 1 are loaded
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quantification.
simultaneously
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a singlesample.
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files, withthethelarger
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were The
run. Thefirst analyzed
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compare phosphopeptides while thethe
second analyzed thethe
combined
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samples from
from phoshpopeptide
phoshpopeptide enrichment
enrichment were wererunrunasasseparate
separate LC/MS/MS
LC/MS/MS runs.
runs. enriched fractions compare phosphopeptides while second analyzed combined
Once thethe
datasets were imported into thethesoftware, thethe
study factors defined in in
Figure flow-through
flow-through and
andwash
wash samples
samples to to
compare
comparechanges
changes in in
protein abundances
protein abundances across
across thethe P
Once datasets were imported into software, study factors defined Figure samples.
1a1awere
were assigned
assigned to to
each
eachsample
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shown in in
Figure
Figure 1b1b above.
above. samples.
L
The third step is is
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The
asaswell
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that includes a Processing
a ProcessingWorkflow
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peptide Phosphopeptide search results
identification, peptide quantification, FDR calculation and PTM site localization and Phosphopeptide search results M
identification, peptide quantification, FDR calculation and PTM site localization and
adds
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Consensus workflow
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that used
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perform protein inference,
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filter The
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thus IMAC
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results
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othercalculations,
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summarize quantification
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results. enrichment
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dataforfor
samples
samples 3955
3955and
and 3956
3956 were
were removed
removed from
from thethe
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analysisforfor
clarity.
clarity. D
The two workflows used forfor
this study are shown in in
Figures 2a-b below. AsAs
a result,
a result,there
therewere
were two
twotechnical
technicalreplicates
replicates forfor
thethe
TiO 2-enriched
TiO phosphopeptides for
The two workflows used this study are shown Figures 2a-b below. 2-enriched phosphopeptides for
each
eachof of
thetheoriginal
originalsamples
samples leading
leadingto to
8 different
8 different quantitative
quantitativecategories
categories asasshown
shown in in T
Processing
Processing Consensus
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242 raw files.
Figure 4 for 242 raw files.Figure 4 shows
Figure 4 shows a screenshot
a screenshot of of
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results of of
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phosphopeptide data.
data.
V

Figure 4. 4.
Figure Identified peptide
Identified peptidegroups
groupsforfor
TiO 2-enriched
TiO phosphopeptides. The fourth
2-enriched phosphopeptides. The fourth
column
columnshows
shows the
themodification
modification and the
and thesite
sitelocalization probability
localization probability calculated
calculatedbyby
ptmRS.
ptmRS.The The“Area
“Areacolumns”
columns” show
showthethepeak
peak area calculated
area calculated forfor
that identified
that identified
FIGURE
FIGURE2. 2.Processing and
Processing consensus
and workflows
consensus forfor
workflows the phosphopeptide
the phosphopeptide phosphopeptide
phosphopeptide across allall
across 8 multidimensional
8 multidimensional LC/MS/MS
LC/MS/MS runs.
runs.The Theselected
selected
analysis.
analysis. peptide is is
peptide about
about20x
20xmore
moreabundant
abundant inin
the hESCs
the hESCscompared
compared toto
the hNSCs
the hNSCs with
with
good biological and technical reproducibility.
good biological and technical reproducibility.
TheTheprocessing
processing workflow
workflow in in
Figure
Figure 2 includes
2 includes Sequest
Sequest HTHTnodes
nodes to to
interpret
interpret both
bothCID
CID
andandETDETD MS/MS
MS/MSandandincludes
includes thethe
Event
EventDetector
Detectorand
andPrecursor
Precursor Ions
IonsAreaAreaDetector
Detector Table 1 shows
Table 1 shows a summary
a summary of of
thethe
number
number of of
PSM’s,
PSM’s,peptides,
peptides, andandphosphopeptides
phosphopeptides
nodes to calculate peak areas for identified peptides. All Sequest HT
nodes to calculate peak areas for identified peptides. All Sequest HT searches used searches used identified across
identified across thethe
4 samples.
4 samples.ToTo find phosphorylation
find phosphorylation sites that
sites are
that unique
are unique to to
thethe
two
two
1010 ppm
ppm precursor
precursormass
mass tolerance,
tolerance, while thethe
while CIDCIDdata used
data used0.60.6
m/zm/zfragment
fragment tolerance
tolerance hESC samples (3955 and 3957), the row filters were selected
hESC samples (3955 and 3957), the row filters were selected to show to show phosphopeptides
phosphopeptides
andandthetheETD
ETDdata
dataused
used1.21.2 m/zm/zfragment
fragment tolerance.
tolerance.AllAll
searches
searches used
used fixed
fixed that appear in one of the two replicates in each case that were not identified
that appear in one of the two replicates in each case that were not identified in the two in the two
carbamidomethylation, variable phosphorylation (S,T,Y), oxidation (M),
carbamidomethylation, variable phosphorylation (S,T,Y), oxidation (M), and pyro-Glu and pyro-Glu hNSC
hNSC samples
samples (3956
(3956 and
and3958).
3958).In In
total, there
total, were
there were811
811unique
unique phosphopeptides
phosphopeptides
(peptide
(peptide N-term Q).Q).Finally,
N-term Finally, thetheptmRS
ptmRS node
node waswasappended
appended to to
thethe
endend of of
thetheworkflow
workflow identified only
identified in in
only thethe
hESC
hESCsamples
samples versus
versus 253
253unique phosphopeptides
unique phosphopeptides identified
identified only in in
only
to to
calculate
calculate modification
modification site localization
site localizationprobability.
probability. thethe
two hNSC samples.
two hNSC samples.
The Consensus
The Consensus workflow is is
workflow used
usedto to
calculate
calculate protein
proteingroups,
groups,filtering peptides
filtering peptides and
and Sample
Sample PSM’s
PSM’s Unique
Unique Unique
Unique Unique
Unique
proteins byby
proteins false discovery
false rate,
discovery and
rate, and rolling upup
rolling thethe
quantification
quantification results from
results individual
from individual peptides
peptides phoshpopeptides
phoshpopeptides phosphopeptides
phosphopeptides
peptide spectral matches (PSM’s) to peptide groups and
peptide spectral matches (PSM’s) to peptide groups and proteins.proteins. (ptmRS isoform
(ptmRS score
isoform score
>60)
>60)
a)a) b)b) hESC
hESC11 38174
38174 7715
7715 5575
5575 4151
4151
hNSC
hNSC11 21987
21987 4562
4562 3191
3191 2388
2388
hESC
hESC22 47338
47338 7835
7835 6223
6223 4526
4526
hNSC
hNSC22 35350
35350 7086
7086 48983677
48983677
TABLE 1. Identification summary for enriched phosphopeptide samples.
TABLE 1. Identification summary for enriched phosphopeptide samples.
For thethe
For unique
unique hESC
hESC phosphopeptides,
phosphopeptides, overrepresentation
overrepresentation analysis
analysisof of
pathways
pathways waswas
performed
performed and
andinsulin
insulinsignaling, MAPK,
signaling, MAPK, ErbB, and
ErbB, andAMPK
AMPK pathways
pathways were
were identified.
identified.For
For
thethe
peptides unique to the hNSC samples, there were several phosphorylation
peptides unique to the hNSC samples, there were several phosphorylation sites sites
detected
detected ononthethe
protein
protein MAP2,
MAP2,which is is
which known
known to to
bebe
involved
involvedin in
neurogenesis,
neurogenesis, SOX-5,
SOX-5,
which
which is is
involved
involvedin in
chondrogenesis,
chondrogenesis, and
and neuron-navigator
neuron-navigator 1. 1.

FIGURE
FIGURE 3. 3.Study factors
Study forfor
factors the enriched
the enriched(a)(a)
phosphopeptide search
phosphopeptide searchand
andthe
the
flow-through
flow-through and wash
and washfractions
fractions (b).
(b).There
Thereare asas
are many columns
many columnsinin
the final
the final
report as are shown in green on this table.
report as are shown in green on this table.

2 Quantitative Analysis of Large Phosphopeptide Datasets Using Proteome Discoverer 2.0

. Some
Some phosphopeptides
phosphopeptides were
were also
also identified
identified in in
allall samples
samples but
but are
are also
also differentially
differentially
e abundant.There
There were Sample
Sample Enrichment Replicate
Enrichment Replicate Protein
Protein groups
groups Peptide
Peptide Groups
Groups
abundant. were 3838 phosphopeptides
phosphopeptides that
that appear
appear toto
bebe up-regulated
up-regulated byby a factor
a factor
ofof
≥2≥2
in in hNSCs
hNSCs while
while there
there were
were 101101 phosphopeptides
phosphopeptides that
that were
were up-regulated
up-regulated in in
hESCs.The
hESCs. The phosphopeptides
phosphopeptides with
with thethe highest
highest differential
differential expression
expression ratios
ratios between
between
the
the hESC
hESC and
and hNSC
hNSC samples
samples are
are shown
shown in in Table
Table 2.2.
d hESC
hESC 11 TiO
TiO 2 2 11 6240
6240 47667
47667
Peptide
Peptide Protein
Protein Ratio
Ratio hESC
hESC 11 TiO
TiO 2 2 22 6303
6303 48105
48105
hNSC/hESC
hNSC/hESC hESC
hESC 11 IMAC
IMAC 11 5854
5854 35924
35924
LKCGSGPVHISGQHLVAVEEDAESEDEEEEDVK
LKCGSGPVHISGQHLVAVEEDAESEDEEEEDVK Rho
Rho GTPase
GTPase activating
activating protein
protein 1717 0.025
0.025
hESC
hESC 11 IMAC
IMAC 22 5991
5991 36990
36990
RPTPNDDTLDEGVGLVHSNIATEHIPSPAK
RPTPNDDTLDEGVGLVHSNIATEHIPSPAK Hydroxymethylglutaryl-CoA
Hydroxymethylglutaryl-CoA synthase
synthase 0.032
0.032
hNSC
hNSC 11 TiO
TiO 2 2 11 7047
7047 54935
54935
MAPTPIPTRSPSDSSTASTPVAEQIER
MAPTPIPTRSPSDSSTASTPVAEQIER Drebrin
Drebrin 0.055
0.055
hNSC
hNSC 11 TiO
TiO 2 2 22 7044
7044 55473
55473
SSMSGLHLVK
SSMSGLHLVK Acetyl-CoA
Acetyl-CoA carboxylase
carboxylase 1 1 0.058
0.058
hNSC
hNSC 11 IMAC
IMAC 11 6551
6551 43585
43585
DMESPTKLDVTLAK
DMESPTKLDVTLAK Microtubule
Microtubule associated
associated protein
protein 4 4 0.068
0.068
or hNSC
hNSC 11 IMAC
IMAC 22 6654
6654 44475
44475
TTRTPEEGGYSYDISEK
TTRTPEEGGYSYDISEK Microtubule
Microtubule associated
associated protein
protein 1B1B 1818
hESC
hESC 22 TiO
TiO 2 2 11 6701
6701 54023
54023
RPASPSSPEHLPATPAESPAQR
RPASPSSPEHLPATPAESPAQR Sin3
Sin3 histone
histone deacetylase
deacetylase corepressor
corepressor 1919
complex component SDS3
hESC
hESC 22 TiO
TiO 2 2 22 6776
6776 54385
54385
complex component SDS3
VALSDDETKETENMR
VALSDDETKETENMR DNA
DNA polymerase
polymerase delta
delta subunit
subunit 3 3 2121
hNSC
hNSC 22 TiO
TiO 2 2 11 6605
6605 50303
50303
RSTQGVTLTDLQEAEK
RSTQGVTLTDLQEAEK Protein
Protein phosphatase
phosphatase 1 regulatory
1 regulatory subunit
subunit 3131
hNSC
hNSC 22 TiO
TiO 2 2 22 6639
6639 50887
50887
12A
12A
TABLE
TABLE 3.3. Summary
Summary ofof protein
protein identifications
identifications forfor each
each sample.
sample.
IEDSEPHIPLIDDTDAEDDAPTKR
IEDSEPHIPLIDDTDAEDDAPTKR Plasma
Plasma membrane
membrane calcium-transporting
calcium-transporting 120
120
ATPase
ATPase 1 1 TheThe table
table ofof protein
protein identifications
identifications andand quantitative
quantitative information
information waswas exported
exported toto Excel.
Excel.
ToTo account
account forfor differences
differences in in the
the injection
injection amounts
amounts forfor each
each sample,
sample, thethe abundances
abundances
TABLE
TABLE 2.2.Phosphopeptides
Phosphopeptides with
with the
the lowest
lowest and
and highest
highest quantitative
quantitative ratios
ratios forfor each
each protein
protein were
were normalized
normalized toto
thethe summed
summed abundance
abundance ofof
allall proteins
proteins in in the
the
between
between hESCs
hESCs and
and hNSCs.
hNSCs. sample.The
sample. The results
results were
were imported
imported into
into ProteinCenter
ProteinCenter and
and profiled.The
profiled. The top
top andand
bottom
bottom clusters
clusters showed
showed overrepresentation
overrepresentation in in hNSCs
hNSCs and
and hESCs
hESCs respectively
respectively (Figure
(Figure
8).8).
Protein
Protein Quantification
Quantification
For
For protein
protein quantification,
quantification, another
another setset
ofof searches
searches were
were performed
performed onon
thethe flow-through
flow-through
and
and wash
wash fractions
fractions from
from the
the phosphopeptide
phosphopeptide enrichment
enrichment steps.The
steps. The same
same workflows
workflows
were
were used
used asas shown
shown in in Figure
Figure 2,2,
butbut phosphorylation
phosphorylation waswas removed
removed asas a variable
a variable
modification
modification from
from the
the Sequest
Sequest HTHT searches
searches andand Protein
Protein FDRFDR threshold
threshold node
node setsettoto
1%1%
h was
was added.For
added. For these
these data,
data, thethe “Proteins”
“Proteins” tab
tab in in the
the final
final results
results show
show thethe average
average ofof
the
the top
top 3 most
3 most abundant
abundant peptides
peptides detected
detected across
across allall
ofofthethe samples
samples asas seen
seen in in Figure
Figure
5 below.
5 below.

FIGURE
FIGURE 8.8.Top
Top two
two clusters
clusters from
from ProteinCenter
ProteinCenter profiling.The
profiling. The upper
upper cluster
cluster
corresponds
corresponds toto proteins
proteins more
more abundant
abundant inin the
the hNSCs
hNSCs whereas
whereas the
the lower
lower cluster
cluster
corresponds
corresponds toto proteins
proteins that
that are
are more
more abundant
abundant inin the
the hESCs.
hESCs.
Some
Some selected
selected proteins
proteins in in cluster
cluster 1 include
1 include several
several ephrin
ephrin receptors,
receptors, PAX-6,
PAX-6, and
and
n frizzled-3
frizzled-3 precursor
precursor allall
ofof which
which are
are involved
involved in in neuronal
neuronal development
development in in animal
animal
models,
models, and
and SOX-2,
SOX-2, which
which is is known
known toto
bebe involved
involved in in stem
stem cell
cell pluripotency
pluripotency and
and
differentiation.Cluster
differentiation. Cluster 2 contains
2 contains dozens
dozens ofof highly
highly up-regulated
up-regulated (≥10x)
(≥10x) proteins
proteins
including
including gamma-synuclein,
gamma-synuclein, CD9CD9 antigen,
antigen, calveolin-1
calveolin-1 isoform
isoform alpha,
alpha, HRAS-like
HRAS-like
suppressor
suppressor 3,3,
andand nocturnin.
nocturnin.

Conclusion
Conclusion
• • Proteome
Proteome Discoverer
Discoverer 2.0
2.0 software
software is is well
well equipped
equipped toto analyze
analyze highly
highly complex
complex
datasets,
datasets, in in this
this case
case a dataset
a dataset with
with well
well over
over 800
800 RAW
RAW data
data files.
files.
Figure
Figure 5.5. Identified
Identified proteins
proteins forfor the
the flow-through
flow-through and
and wash
wash fractions
fractions across
across the
the
samples.The The “Areas” table shows the average • • The
The study
study management
management feature
feature waswas used
used toto produce
produce peak
peak area
area quantification
quantification
samples. “Areas” table shows the average ofof the
the peak
peak areas
areas ofof the
the top
top 33 values
identified peptides from that protein values forfor phosphopeptides
phosphopeptides and
and the
the proteins
proteins from
from the
the flow-through
flow-through and
and wash
wash
identified peptides from that protein forfor each
each sample.
sample. fractions from TiO
fractions from TiO 2 and
2 and
IMAC
IMAC enrichment.
enrichment.
AA total
total ofof 10682
10682 unique
unique proteins
proteins were
were identified
identified across
across allall
ofof the
the samples.Table
samples. Table33
shows the proteins identified • • The
The precursor
precursor ionion quantification
quantification can
can bebe used
used toto find
find differentially
differentially expressed
expressed
shows the proteins identified forfor each
each ofofthethe samples,
samples, with
with roughly
roughly 6500-7000
6500-7000 proteins
proteins phosphopeptides
identified phosphopeptides asas well
well asas proteins
proteins between
between thethe hESC
hESC and
and hNSC
hNSC samples.
samples.
identified in in each
each sample.Row
sample. Row filters
filters were
were applied
applied toto show
show only
only those
those proteins
proteins
identified
identified in in the
the various
various hESC
hESC samples,
samples, with
with a total
a total ofof 285
285 proteins
proteins with
with twotwo oror more
more • • ProteinCenter
ProteinCenter aids
aids in in the
the biological
biological interpretation
interpretation ofof
thethe Proteome
Proteome Discoverer
Discoverer
unique
unique peptides
peptides identified
identified in in
atat least
least oneone ofof
thethe hESC
hESC samples
samples but
but none
none ofof
thethe hNSC
hNSC software
software results.
results.
samples
samples (data
(data notnot shown).This
shown). This includes
includes proteins
proteins such
such asas cadherin-3,
cadherin-3, PR PR domain
domain zinc
zinc
finger
finger protein
protein 14,14, Tyrosine-protein
Tyrosine-protein kinase
kinase Lck,
Lck, and
and Oct4.A A
Oct4. second
second setset
ofof filters
filters were
were
used
used toto
showshow proteins
proteins that
that only
only appear
appear in in
oneoneoror more
more hNSC
hNSC samples,
samples, which
which
produced
produced a list
a list ofof458458 proteins
proteins with
with atat least
least 2 peptides.Selected
2 peptides. Selected proteins
proteins include
include © 2015
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Thermo Fisher
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Scientific Inc.Inc.
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reserved. AllAll trademarks
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others.
precursor,contactin-associated
precursor, contactin-associated protein
protein 1 precursor,
1 precursor, and
and Spondin-1.
Spondin-1.

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