Quantitative phosphoproteomic analysis of prion-infected neuronal cells
Cell Communication and Signaling
Quantitative phosphoproteomic analysis of prion-infected neuronal cells
Wibke Wagner 0
Paul Ajuh 2
Johannes Lwer 0
Silja Wessler 0 1
0 Paul Ehrlich Institute , Paul Ehrlich-Strae 51-59, D-63225 Langen , Germany
1 Division of Microbiology, Paris-Lodron University , Salzburg , Austria
2 Dundee Cell Products Ltd , James Lindsay Place, Dundee Technopole Dundee, DD1 5JJ , UK
Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal diseases associated with the conversion of the cellular prion protein (PrPC) to the abnormal prion protein (PrPSc). Since the molecular mechanisms in pathogenesis are widely unclear, we analyzed the global phospho-proteome and detected a differential pattern of tyrosine- and threonine phosphorylated proteins in PrPSc-replicating and pentosan polysulfate (PPS)-rescued N2a cells in two-dimensional gel electrophoresis. To quantify phosphorylated proteins, we performed a SILAC (stable isotope labeling by amino acids in cell culture) analysis and identified 105 proteins, which showed a regulated phosphorylation upon PrPSc infection. Among those proteins, we validated the dephosphorylation of stathmin and Cdc2 and the induced phosphorylation of cofilin in PrPSc-infected N2a cells in Western blot analyses. Our analysis showed for the first time a differentially regulated phospho-proteome in PrPSc infection, which could contribute to the establishment of novel protein markers and to the development of novel therapeutic intervention strategies in targeting prion-associated disease.
Transmissible spongiform encephalopathies (TSEs) are
fatal neurodegenerative diseases occurring in many
different host species including humans, which develop e.g.
Creutzfeld Jacob disease (sCJD) . The development of
TSEs is associated with the self-propagating conversion
of the normal host cellular prion protein (PrPC) into the
abnormal protease-resistant isoform (PrPSc or PrPres) in
an autocatalytic manner . PrPSc plays a key role as an
infectious agent in certain degenerative diseases of the
central nervous system .
The cellular functions of PrPC and PrPSc still remain
enigmatic. The cellular prion protein can be variably
glycosylated at two N-glycosylation sites and is C-terminally
attached to the cell surface by a glycosyl
phosphatidylinositol (GPI) anchor. GPI-anchored proteins are found in
lipid rafts, highly cholesterol- and glycolipid-enriched
membrane domains associated with a large number of
signaling molecules such as G-protein-coupled receptors
and protein kinases suggesting that signaling
transduction pathways might play a role in TSEs . Hence,
previous publications described a functional role of PrPC
as a signaling molecule with major findings indicating
that PrPC interacts with and activates Src family kinases
[5-7]. Increased levels of active Src kinases in
scrapieinfected cells then led to the activation of downstream
signal transduction pathways . Recently, activation of
the JAK-STAT signaling pathway in astrocytes of
scrapie-infected brains was observed underlining that signal
transduction pathways may play pivotal roles in prion
pathogenesis . Interestingly, it was demonstrated that
inhibition of the non-receptor tyrosine kinase c-Abl
strongly activates the lysosomal degradation of PrPSc
. These data indicate that specific interference with
cellular signaling pathways could represent a novel
strategy in treatment of TSEs.
We have performed a quantitative analysis of the
phospho-proteome to obtain a global insight into deregulated
signal transduction pathways in scrapie-infected neuronal
cells. We analyzed tyrosine- and threonine-phosphorylated
proteins in the murine neuroblastoma cell line N2a58/22L,
which were infected with the PrPSc strain 22L . We
have treated N2a58/22L cells with pentosan polysulfate
(PPS), a known inhibitor of 22L PrPSc replication in N2a
cells , resulting in the PrPSc-rescued cell line N2a58#
which served as an uninfected control. Successful rescue
from PrPSc was demonstrated in the colony assay as
reflected by the absence of proteinase K (PK)-resistant
PrPSc in N2a58# cells after PPS treatment (Figure 1A).
Figure 1 Differentially phosphorylated proteins in PrPSc-positive and -negative N2a cells. (A) PrPres-positive N2a58/22L cells were treated
with pentosan polysulfate (PPS) to obtain PrPres-negative N2a58# cells. Successful PPS treatment was validated in a colony assay. Cells were
grown to confluence on cover slips and directly lysed on nitrocellulose. Where indicated 20 g/ml proteinase K (PK) was added followed by the
detection of PrP expression using the 6H4 monoclonal antibody. In non-treated cells (-), PrP was detected in both, cured and infected N2a cells.
Upon digestion with PK (+), PrPres was only observed in N2a58/22L cells. (B) Equal amounts of protein lysates were incubated with 20 g/ml PK
or left untreated. PrP was detected with the 8H4 monoclonal antibody showing the typical migration pattern of PrP and PrPres in infected and
PPS-treated N2a58# cells. In parallel, lysates were incubated with PK to visualize PK-resistant PrPres in N2a58/22L. (C) 150 g of N2a58# or
prioninfected N2a58/22L cell lysates were separated by two-dimensional gel electrophoresis followed either by Coomassie staining or immunoblotting
for detection of tyrosine- and threonine-phosphorylated proteins. Black asterisks indicate changed intensities of protein phosphorylation.
PrPSc replication and the effect of PPS-treatment were
further studied in an immunoblot. After PK digestion,
PrPSc replication was only observed in N2a58/22L cells
(Figure 1B, lanes 2 and 4). Compared to 22L-infected
N2a58/22L cells, PPS-treated N2a58# cells showed a
different glycosylation profile as expected for PrPC [13-15].
The glycosylation pattern of PrPC in N2a58# cells
displayed high amounts of di- and mono-glycosylated PrPC,
whereas in N2a58/22L cells predominantly mono- and
non-glycosylated PrPSc was detected (Figure 1B, lanes 1
and 3). Altogether, PPS treatment of N2a58/22L cells
successfully abolished PrPSc formation in N2a58# cells, which
served as a non-infected control cell line in our study.
To analyze differentially phosphorylated proteins in
N2a58/22L cells in comparison to N2a58# cells, we
separated equal protein amounts by two-dimensional gel
electrophoresis. Gels were stained with Coomassie Blue
to demonstrate equal protein amounts in N2a58/22L
and N2a58# cells (Figure 1C, left panels). In parallel,
gels were blotted onto membranes and incubated
with phospho-specific antibodies to detect
tyrosine(Figure 1C, middle panels) or threonine-phosphorylated
proteins (Figure 1C, right panels). Interestingly,
considerable differences in phosphorylation patterns were
observed (Figure 1C, asterisks), while other
phosphorylated proteins were not changed in N2a58/22L and
N2a58# cells (Figure 1C). These data imply differentially
regulated phosphoproteins in response to 22L infection
of neuronal cells.
Generally, global detection of phosphorylated proteins
is still challenging, as antisera often recognize
phosphorylated residues dependent on the surrounding sequence.
For a general detection of proteins post-translationally
phosphorylated at those sites, we performed a SILAC
analysis allowing the identification and relative
quantification of differential phosphoprotein regulation.
Therefore, N2a58# cells were grown in light isotope containing
and N2a58/22L cells in heavy isotope containing
medium. Equal amounts of protein lysates were mixed,
separated by gel electrophoresis, trypsinized and followed
by enrichment of phosphoproteins, which were then
analyzed by mass spectrometry. We identified 109 different
phosphoproteins of which 105 were also quantified
(Tables 1 and 2). We observed 75 proteins with a ratio of
identified peptides in N2a58/22L versus N2a58#
cells ranging from 0.46 to 0.99 (Table 1). Conversely, 30
phosphoproteins showed a ratio between 1.01 and 1.79
(Table 2). We defined proteins exhibiting a ratio < 0.70
as dephosphorylated proteins and proteins with ratios
between 0.70 and 1.40 as proteins, whose
phosphorylation was not altered in 22L-infected N2a58/22L cells.
Ratios > 1.40 were considered as proteins whose
phosphorylation increased upon Scrapie infection.
Among quantified phosphoproteins, we then
considered specific phosphosites in selected target proteins,
such as Cdc2, stathmin, and cofilin as analyzed by
massspectrometry (Table 3). An increase of cofilinS3
phosphorylation in N2a58/22L cells was suggested by a ratio
1.63, while the amount of the two tyrosine
phosphorylation sites (Y15, Y160) in Cdc2 were decreased upon 22L
infection. Stathmin phosphopeptides containing serine
38 were increased, whereas the amount of stathmin
phosphopeptides harboring serine 25 in N2a58/22L cells
was significantly lower (Table 3).
To validate the results obtained in the SILAC
phosphoproteomic analysis we performed Western blots for
cofilin 1, Cdc2, and stathmin using antibodies for the
detection of specific phosphosites. As predicted by the
SILAC analysis, cofilin 1 phosphorylation was
significantly induced in Scrapie-infected N2a58/22L cells
compared to PPS-treated N2a58# cells (Figure 2, left panels).
Cofilin represents a potent regulator of the actin
filaments, which is controlled by phosphorylation of serine
3 mediated through the LIM-kinase 1 (LIMK-1) in vitro
and in vivo . These data support previous studies
indicating a direct interaction of PrPSc with cofilin .
Together with our finding that phosphorylation of
cofilin is induced in PrPSc-infected neuronal cells; the
results indicate a significant role for the protein in
neurodegeneration processes. Stathmin acts as an important
regulatory protein of microtubule dynamics, which can
be directly targeted by Cdc2 . In our analysis, we
showed that stathminS38 phosphorylation was decreased
(Figure 2, middle panels), which correlates with the
inactivation of Cdc2 in N2a58/22L cells (Figure 2, right
panels) implying that there is a functional interaction.
Cdc2 is a crucial kinase in starting M phase events
during the cell cycle progression and regulates important
mitotic structure changes, including nuclear envelope
breakdown and spindle assembly .
Dephosphorylation of stathminS38 led to an inhibition of cells at G2/M
phase, lack of spindle assembly, and growth inhibition
[20,21]. Together with the finding that the prion gene is
transcriptionally activated in the G1 phase in confluent
and terminally differentiated cells , we assume that
control of the cell cycle might be important in prion
Aberrant signal transduction pathways are implicated
in many diseases. However, perturbations in
phosphorylation-based signaling networks are typically studied in a
hypothesis-driven approach. In this study, we performed
the first global analysis of the phosphoproteome of
scrapie-infected neuronal cells, since the knowledge of
PrPdependent deregulation of the signalling network is
poor. SILAC provides a powerful and accurate technique
for relative proteome-wide quantification by
massspectrometry. Its versatility has been demonstrated by a
wide range of applications, especially for intracellular
signal transduction pathways [23-25]. Since we applied
SILAC for the quantitative detection of the
phosphoproteome in scrapie-infected neuroblastoma cells, we found
105 different phosphoproteins. Among identified
proteins, we validated the regulated phosphorylation of
cofilin, stathmin and Cdc2 indicating that the identification
of phosphoproteins in scrapie-infected neuronal cells by
SILAC is reliable. Future work is necessary to determine
whether the identified novel phosphoproteins are
involved in prion diseases and if they probably represent
sensitive and specific biomarkers for diagnosis or
therapeutic intervention strategies.
N2a58/22L cells have been described previously  and
were kindly provided by Prof. Schtzl (LMU, Munich).
Cells were cultured in DMEM containing 10% FCS and
4 mM L-glutamine at 37C. Cells were treated with
5 g/ml pentosan polysulfate (Cartrophen Vet, A.
Albrecht GmbH + Co. KG, Germany) for two passages,
resulting in a stable rescued cell line for more than 15
passages (N2a58# cells). Cell lysates were prepared by
scraping cells in lysis buffer containing 150 mM NaCl,
0.5% Triton X-100, 0.5% DOC, 50 mM Tris pH 7.5, 1
mM Na-vanadate, 1 mM Na-molybdate, 20 mM NaF,
10 mM NaPP, 20 mM -glycerophosphat, 1 protease
inhibitor cocktail (Roche, Mannheim, Germany). For
digestion with proteinase K (PK) 80 g protein were
treated with 20 g/ml PK for 30 min at 37C. PK
digestion was stopped by addition of laemmli sample buffer
and protein denaturation at 95C for 7 min.
The colony assay was performed as previously described
with minor modifications . In brief, cells were grown
on glass cover slips to confluence using a 24 well plate.
The cell layer was soaked in lysis buffer (150 mM NaCl,
No. Uniprot Protein Names Ratioa Peptb sequence PEPc
1 P43276 Histone H1.5 0.46237 1 13.9
2 P30681 High mobility group protein B2 0.48683 2 11
3 P11440 Cell division control protein 2 homolog 0.49428 5 7.7
4 P97310 DNA replication licensing factor MCM2 0.54657 1 2.4
O70251 Elongation factor 1-beta
Q61656 Probable ATP-dependent RNA helicase DDX5
P09411 Phosphoglycerate kinase 1
P48962 ADP/ATP translocase 1
Q9D8N0 Elongation factor 1-gamma
P49312-2 Heterogeneous nuclear ribonucleoprotein A1
Q9CZM2 60S ribosomal protein L15
P97855 Ras GTPase-activating protein-binding
Q9EQU5-1 Protein SET
Q7TPV4 Myb-binding protein 1A
P80318 T-complex protein 1 subunit gamma
P25444 40S ribosomal protein S2
P10126 Elongation factor 1-alpha 1
P61979-2 Heterogeneous nuclear ribonucleoprotein K
P07901 Heat shock protein HSP 90-alpha
Rab GDP dissociation inhibitor beta
26S protease regulatory subunit 6B 0.81795 2
Heterogeneous nuclear ribonucleoprotein A/B 0.82349 3
40S ribosomal protein SA;Laminin receptor 1 0.8261 7
Actin, alpha skeletal muscle 0.83457 10
T-complex protein 1 subunit beta 0.83579 2
Proliferation-associated protein 2G4 0.84048 2
T-complex protein 1 subunit alpha B 0.84687 4
Calnexin 0.85134 1
Putative uncharacterized protein;26S protease 0.85207 3
regulatory subunit 6A
Heat shock cognate 71 kDa protein 0.85947 9
Nucleoside diphosphate kinase B 0.86107 4
40S ribosomal protein S7 0.86306 3
T-complex protein 1 subunit delta 0.86423 1
Heat shock protein 84b 0.8654 4
78 kDa glucose-regulated protein
0.5% Triton X-100, 0.5% DOC, 50 mM Tris pH 7.5) on a
nitrocellulose membrane. After drying for 30 min at
room temperature, the membrane was incubated in lysis
buffer containing 5 g/ml proteinase K (PK) for 90 min
at 37C, rinsed twice with water, and incubated in 2 mM
PMSF for 10 min. The membrane was shaken in 3 M
guanidinium thiocyanate, 10 mM Tris-HCl (pH 8.0) for
10 min, followed by rinsing five times with water. 5%
nonfat dry milk in TBS-T was used for blocking for 1 h
at room temperature. PrP was detected using an anti-PrP
Table 2 Proteins exhibiting increased phosphorylation in N2a58/22L cells
No. Uniprot Protein Names Ratioa Pept.b Sequence PEPc
P80316 T-complex protein 1 subunit epsilon 1.017 1
Q9CX22 Putative uncharacterized protein; 1.0173 5
P32067 Lupus La protein homolog 1.02 2
A6ZI44 Fructose-bisphosphate aldolase 1.0328 8
P35700 Peroxiredoxin-1 1.0342 6
P05202 Aspartate aminotransferase, 1.0344 1
Q3TFD0 Serine hydroxymethyltransferase 1.0425 3
P63101 14-3-3 protein zeta/delta 1.0478 19
Q9CZ30-1 Obg-like ATPase 1 1.0606 3
P06745 Glucose-6-phosphate isomerase 1.0652 3
Q71H74 Collapsin response mediator protein 1.0688 5
Q6P5F9 Exportin-1 1.0703 2
Q01853 Transitional endoplasmic reticulum 1.0708 2
L-lactate dehydrogenase 1.0746 4
Endoplasmin 1.08 1
Ubc protein;Ubiquitin 1.0848 17
Mtap1b protein 1.0871 2
Peroxiredoxin-2 1.0934 2
Transgelin-2 1.106 2
Pyruvate kinase isozymes M1/M2 1.1081 3
Peroxiredoxin-6 1.1204 5
Alpha-enolase 1.128 11
Stathmin 1.1422 4
Tubulin beta-6 chain 1.1427 1
Stress-induced-phosphoprotein 1 1.1499 1
Pyruvate kinase isozymes M1/M2 1.2958 1
Farnesyl pyrophosphate synthetase 1.3534 1
Stress-70 protein, mitochondrial 1.357 2
Annexin A6 1.6252 4
D-3-phosphoglycerate dehydrogenase 1.7927 3
8.63E-18 protein folding
3.72E-41 cytoskeleton; protein phosphorylation
2.74E-20 nuclear export; centrosome duplication
2.97E-05 apoptosis; retrograde protein transport
a. Ratio of N2a58/22L vs. N2a58# cells
b. Number of identified peptides
c. posterior error probability (PEP) estimates the probability of wrong assignment of a spectrum to a peptide sequence
antibody 6H4 (Prionics) and a HRP-conjugated sheep
anti-mouse antibody (GE Healthcare).
SDS-PAGE and Western Blot
Proteins were separated by 12% SDS-PAGE and
transferred to polyvinylidene difluoride membranes (PVDF,
Millipore) by semidry blotting. PrP was detected using
the PrP-specific mouse mAb 8H4 (Alicon AG). For
validation of phosphorylated proteins anti-phospho-stathmin
(Ser38) (#3426, Cell Signaling Technology),
anti-phospho-cdc2 (Tyr15) (#4539, Cell Signaling Technology),
and anti-phospho-cofilin (Ser3) antibodies (#3313, Cell
Signaling Technology) were used. Antibodies recognizing
Table 3 Identified phosphorylation sites
Protein names Ratio Phosphosite Ratio
(total)a (specific phospho-site)b
Cdc2 0.49428 Y15 0.43086
stathmin 1.1422 S25 1.2155
cofilin 1.0173 S3 1.6328
a. Ratio of phosphorylation N2a58/22L vs. N2a58# cells
b. Ratio of phospho sites in N2a58/22L and N2a58# cells
Figure 2 Specific regulation of cofilin, Cdc2, and stathmin phosphorylation in scrapie-infected neuronal cells. Cell lysates of N2a58# and
22L-infected N2a58/22L cells were analyzed by Western blot using phospho-specific antibodies to detect p-cofilinS3, p-cdc2Y15, and p-stathminS38
(left panels). As loading controls, equal amounts of cofilin, Cdc2 and stathmin were shown. Quantification of intensities of phosphorylation signal
was performed by normalizing the corresponding loading control (* p < 0.05) (right panels).
stathmin (#3352), cdc2 (#9112) and cofilin (#3312) were
also obtained from Cell Signaling Technology.
Two dimensional gel electrophoresis
For 2D electrophoresis 150 g protein of cell lysates were
purified by trichloroacetic acid precipitation and
re-suspended in DeStreak Rehydration Solution (Amersham
Biosciences) containing 0.5% Bio-Lyte pH3-10 (Bio-Rad
Laboratories GmbH, Mnchen). The isoelectric focusing
was run on IPG strips with a non-linear pH range of 3-10
and a length of 7 cm (Bio-Rad) using the ZOOM
IPGRunnersystem from Invitrogen. After focussing
strips were equilibrated in 50 mM Tris, 1 mM Urea, 30%
Glycerin, 2% SDS, 1% DTT for 25 min and in 50 mM
Tris, 1 mM Urea, 30% Glycerin, 2% SDS, 5% Iodacetamid
for 25 min. Strips were then separated in 10% SDS-PAGE
gels in the second dimension and analyzed by Coomassie
staining or immunoblotting using an
anti-phospho-tyrosine (sc-7020, Santa Cruz) or an anti-phospho-threonine
antibody (#9381, Cell Signaling Technology).
SILAC phosphoproteomics analysis
SILAC ready-to-use cell culture media and dialyzed FBS
were obtained from Dundee Cell Products Ltd, UK.
While N2a58# cells were cultured in control SILAC
DMEM media containing unlabelled arginine and lysine
amino acids (R0K0), N2a58/22L cells were cultured in
ready-to-use SILAC DMEM medium containing 13C
labeled arginine and lysine amino acids (R6K6) for seven
cell division cycles. After preparation of cell lysates and
measurement of protein concentration, lysates of
N2a58# and N2a58/22L cells were mixed in a ratio 1:1.
Each sample was reduced in SDS PAGE loading buffer
containing 10 mM DTT and alkylated in 50 mM
iodoacetamide prior to separation by one-dimensional
SDSPAGE (4-12% Bis-Tris Novex mini-gel, Invitrogen) and
visualization by colloidal Coomassie staining (Novex,
Invitrogen). The entire protein gel lane was excised and
cut into 10 gel slices each. Every gel slice was subjected
to in-gel digestion with trypsin . The resulting
tryptic peptides were extracted by 1% formic acid,
acetonitrile, lyophilized in a speedvac (Helena Biosciences).
The lyophilized peptides above were resuspended in 5%
acetic acid (binding buffer) and phosphopeptide
enrichment was carried out using immobilized metal ion
affinity chromatography (IMAC). Immobilized gallium in
the Pierce Ga-IDA Phosphopeptide Enrichment Kit was
used to enrich for phosphopeptides prior to MS/MS
analysis according to the manufacturers instructions
Trypsin digested peptides were separated using an
Ultimate U3000 (Dionex Corporation) nanoflow LC-system
consisting of a solvent degasser, micro and nanoflow
pumps, flow control module, UV detector and a
thermostated autosampler. 10 l of sample (a total of 2 g)
was loaded with a constant flow of 20 l/min onto a
PepMap C18 trap column (0.3 mm id 5 mm, Dionex
Corporation). After trap enrichment peptides were
eluted off onto a PepMap C18 nano column (75 m
15 cm, Dionex Corporation) with a linear gradient of
5-35% solvent B (90% acetonitrile with 0.1% formic acid)
over 65 minutes with a constant flow of 300 nl/min.
The HPLC system was coupled to a LTQ Orbitrap XL
(Thermo Fisher Scientific Inc) via a nano ES ion source
(Proxeon Biosystems). The spray voltage was set to
1.2 kV and the temperature of the heated capillary was
set to 200C. Full scan MS survey spectra (m/z
3351800) in profile mode were acquired in the Orbitrap
with a resolution of 60,000 after accumulation of
500,000 ions. The five most intense peptide ions from
the preview scan in the Orbitrap were fragmented by
collision induced dissociation (normalised collision
energy 35%, activation Q 0.250 and activation time
30 ms) in the LTQ after the accumulation of 10,000
ions. Maximal filling times were 1,000 ms for the full
scans and 150 ms for the MS/MS scans. Precursor ion
charge state screening was enabled and all unassigned
charge states as well as singly charged species were
rejected. The dynamic exclusion list was restricted to a
maximum of 500 entries with a maximum retention
period of 90 seconds and a relative mass window of
10 ppm. The lock mass option was enabled for survey
scans to improve mass accuracy . Data were
acquired using the Xcalibur software.
Quantification and Bioinformatic Analysis
Quantification was performed with MaxQuant version
220.127.116.11 , and was based on two-dimensional
centroid of the isotope clusters within each SILAC pair.
To minimize the effect of outliers, protein ratios were
calculated as the median of all SILAC pair ratios that
belonged to peptides contained in the protein. The
percentage variability of the quantitation was defined
as the standard deviation of the natural logarithm of
all ratios used for obtaining the protein ratio
multiplied by a constant factor 100.
The generation of peak list, SILAC- and extracted ion
current-based quantitation, calculated posterior error
probability, and false discovery rate based on search
engine results, peptide to protein group assembly, and
data filtration and presentation was carried out using
MaxQuant. The derived peak list was searched with the
Mascot search engine (version 2.1.04; Matrix Science,
London, UK) against a concatenated database combining
80,412 proteins from International Protein Index (IPI)
human protein database version 3.6 (forward database),
and the reversed sequences of all proteins (reverse
database). Alternatively, database searches were done using
Mascot (Matrix Science) as the database search engine
and the results saved as a peptide summary before
quantification using MSQuant http://msquant.sourceforge.
net/. Parameters allowed included up to three missed
cleavages and three labeled amino acids (arginine and
lysine). Initial mass deviation of precursor ion and
fragment ions were up to 7 ppm and 0.5 Da, respectively.
The minimum required peptide length was set to 6
amino acids. To pass statistical evaluation, posterior
error probability (PEP) for peptide identification (MS/MS
spectra) should be below or equal to 0.1. The required
false positive rate (FPR) was set to 5% at the peptide
level. False positive rates or PEP for peptides were
calculated by recording the Mascot score and peptide
sequence length-dependent histograms of forward and
reverse hits separately and then using Bayes theorem in
deriving the probability of a false identification for a
given top scoring peptide. At the protein level, the false
discovery rate (FDR) was calculated as the product of the
PEP of a proteins peptides where only peptides with
distinct sequences were taken into account. If a group of
identified peptide sequences belong to multiple proteins
and these proteins cannot be distinguished, with no
unique peptide reported, these proteins are reported as a
protein group in MaxQuant. Proteins were quantified if
at least one MaxQuant-quantifiable SILAC pair was
present. Identification was set to a false discovery rate of 1%
with a minimum of two quantifiable peptides. The set
value for FPR/PEP at the peptide level ensures that the
worst identified peptide has a probability of 0.05 of being
false; and proteins are sorted by the product of the false
positive rates of their peptides where only peptides with
distinct sequences are recognized. During the search,
proteins are successively included starting with the
bestidentified ones until a false discovery rate of 1% is
reached; an estimation based on the fraction of reverse
Enzyme specificity was set to trypsin allowing for
cleavage of N-terminal to proline and between aspartic acid
and proline. Carbamidomethylation of cysteine was
searched as a fixed modification, whereas N-acetyl
protein, oxidation of methionine and phosphorylation of
serine, threonine and tyrosine were searched as variable
WW carried out the experimental work, drafted and wrote the manuscript.
PA performed and interpreted the SILAC analysis. JL participated in the
design of the study. SW conceived of the study, and participated in its
design and coordination and wrote the manuscript. All authors read and
approved the final manuscript.
The authors declare that they have no competing interests.
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