an
lin
re
fra
an
ge
(NE), chromatin-bound (CB) and pellet (PE) extracts. Each subcellular protein fraction
was briefly separated by SDS-PAGE before in-gel reduction, alkylation and digestion
with trypsin. Peptides from each fraction were labeled with Thermo Scientific™
TMT6plex™ reagents and fractionated by high pH reversed phase spin columns to
generate eight fractions for LC/MS analysis. Label-free peptides from each fraction
were also analyzed by LC/MS. Western blotting using antibodies against HSP90,
EGFR, VDAC, calreticulin, SP1, histone 3, HDAC2, vimentin and cytokeratin 18 was
also performed.
A
Liquid Chromatography
Samples were separated by RP-HPLC using a Thermo Scientific™ Dionex™ Ultimate
3000 system connected to a Thermo Scientific™ EASY-Spray™ column, 15 cm × 75
µm or a Thermo Scientific™ Acclaim™ PepMap™ C18 column, 50 cm × 75 µm over a
3 hr. 5-30% gradient (A: water, 0.1% formic acid; B: acetonitrile, 0.1% formic acid) at
300 nL/min flow rate.
Mass Spectrometry
Spectra were acquired using an Orbitrap Fusion mass spectrometer using top speed
FTMS full scan at 120,000 and FT MS2 HCD 30K resolution or IT MS2 CID and FT
MS3
HCD
usingand
10 fragments
the MS2 spectra).1
Opperman,
John
C.(SPS
Rogers
Barbara from
Kaboord
Poster Note 6445 8
Protein Fractionation by Subcellular
Location to Enhance Proteomic
Coverage of Cultured Cells
Protein fractionation by subcellular location to enhance proteomic
Haiyan Wu, Ryan Bomgarden, Scott Meier, Kay
Haiyan Wu, Ryan
Bomgarden, Scott Meier,Data
Kay
Opperman, John C. Rogers and BarbaB
Analysis
Thermo Fisher Scientific, Rockford, IL, USA
Thermo Fisher
Scientific, Rockford,
IL, USA location to enhance proteomic
Protein
fractionation
by subcellular
Spectral data files were analyzed using Thermo Scientific™ Proteome Discoverer™
1.4 software using the SEQUEST®HT search engine with a precursor mass tolerance
of 10 ppm and fragment mass tolerance of 0.02 Da. Carbamidomethylation (+57.021
Da) for cysteine and TMT isobaric labeling (+229.162 Da) for lysine and N-terminus
residues were treated as static modifications with variable methionine oxidation
(+15.996 Da). Data was searched against a Swiss-Prot® human database with a 1%
FDR criteria using Percolator.
Haiyan Wu, Ryan Bomgarden, Scott Meier, Kay Opperman, John C. Rogers and Barba
Proteome Discoverer 1.4 software was used to calculate TMT reporter ratios with mass
Thermo
Overview Fisher Scientific, Rockford, IL, USA
Results
tolerance ±10 ppm without applying the isotopic correction factors. A protein ratio was
Purpose: To improve protein identification and identify co-localizing proteins through
differential detergent subcellular fractionation.
Methods: HeLa cells were fractionated using the Thermo Scientific™ Pierce™
Subcellular Protein Fractionation Kit for Cultured Cells to segregate and enrich proteins
from cytoplasmic, membrane, nuclear soluble, chromatin-bound and cytoskeleton
compartments. Samples from each fraction were labeled with Thermo Scientific™
TMT™ reagents and analyzed/quantified by LC-MS using a Thermo Scientific™
Purpose: To improve protein identification and identify co-localizing proteins through
Orbitrap™ Fusion™ Tribrid™ mass spectrometer.
differential detergent subcellular fractionation.
Results: Protein fractionation reproducibly separated proteins by cellular compartment
Methods: HeLa cells were fractionated using the Thermo Scientific™ Pierce™
and/or detergent solubility resulting in increased proteome coverage.
Subcellular Protein Fractionation Kit for Cultured Cells to segregate and enrich proteins
from cytoplasmic, membrane, nuclear soluble, chromatin-bound and cytoskeleton
compartments. Samples from each fraction were labeled with Thermo Scientific™
TMT™ reagents and analyzed/quantified by LC-MS using a Thermo Scientific™
Protein localization
traditionally
studied
microscopically using immunofluorescence
Orbitrap™
Fusion™isTribrid™
mass
spectrometer.
or fluorescent protein fusions. Biochemical isolation of various cellular compartments or
Results:
fractionation
reproducibly
separated
proteins
by cellular followed
compartment
organellesProtein
typically
utilizes density
gradients
or differential
centrifugation
by
and/or
solubility
resulting in increased
proteome
coverage.
westerndetergent
blotting of
1D- or 2D-polyacrylamide
gels.
A limitation
of these approaches is
the need for detection antibodies against specific proteins of interest and marker
proteins. Alternatively, subcellular proteomic analysis by mass spectrometry not only
provides information about a specific protein’s subcellular location, but also reveals
global protein
expression
patternsstudied
in eachmicroscopically
specific subcellular
Here, we
Protein
localization
is traditionally
usingcompartment.
immunofluorescence
demonstrate
biochemical,
detergent-based
proteinoffractionation
approach
combinedor
or
fluorescentaprotein
fusions.
Biochemical isolation
various cellular
compartments
with high resolution
mass spectrometry
to examine
a broader
spectrum offollowed
proteinsbyin
organelles
typically utilizes
density gradients
or differential
centrifugation
cytoplasmic,
membrane,
chromatin-bound,
cytoskeleton
derived
western
blotting
of 1D- ornuclear,
2D-polyacrylamide
gels. A and
limitation
of thesefractions
approaches
is
fromneed
HeLa
the
forcells.
detection antibodies against specific proteins of interest and marker
proteins. Alternatively, subcellular proteomic analysis by mass spectrometry not only
provides information about a specific protein’s subcellular location, but also reveals
global protein expression patterns in each specific subcellular compartment. Here, we
demonstrate
a biochemical, detergent-based protein fractionation approach combined
Sample Preparation
with high resolution mass spectrometry to examine a broader spectrum of proteins in
HeLa cells were
lyzed using
50mM
HEPES pH 8, 1%SDS
with sonication
to generate
cytoplasmic,
membrane,
nuclear,
chromatin-bound,
and cytoskeleton
fractions
deriveda
wholeHeLa
cell extract
from
cells. (WCE) or processed using Pierce Subcellular Protein Fractionation
Kit for Cultured Cells to generate cytoplasmic (CE), membrane (ME), nuclear soluble
(NE), chromatin-bound (CB) and pellet (PE) extracts. Each subcellular protein fraction
was briefly separated by SDS-PAGE before in-gel reduction, alkylation and digestion
with trypsin. Peptides from each fraction were labeled with Thermo Scientific™
Sample Preparation
TMT6plex™ reagents and fractionated by high pH reversed phase spin columns to
generate
for LC/MS
Label-free
peptides
from eachtofraction
HeLa
cellseight
werefractions
lyzed using
50mM analysis.
HEPES pH
8, 1%SDS
with sonication
generate a
were also
by LC/MS.
Westernusing
blotting
using
antibodiesProtein
againstFractionation
HSP90,
whole
cell analyzed
extract (WCE)
or processed
Pierce
Subcellular
EGFR,
VDAC, calreticulin,
SP1, histone
3, HDAC2,
vimentin and
cytokeratin
was
Kit
for Cultured
Cells to generate
cytoplasmic
(CE), membrane
(ME),
nuclear 18
soluble
also performed.
(NE),
chromatin-bound (CB) and pellet (PE) extracts. Each subcellular protein fraction
was briefly separated by SDS-PAGE before in-gel reduction, alkylation and digestion
Liquid Chromatography
with trypsin. Peptides from each fraction were labeled with Thermo Scientific™
Samples were
separated
RP-HPLC by
using
Thermo
Scientific™
Dionex™
Ultimate
TMT6plex™
reagents
andby
fractionated
higha pH
reversed
phase spin
columns
to
3000 system
to aLC/MS
Thermo
Scientific™
EASY-Spray™
column,
15fraction
cm × 75
generate
eightconnected
fractions for
analysis.
Label-free
peptides from
each
µm oralso
a Thermo
Scientific™
Acclaim™
C18antibodies
column, 50
cm × 75
µm over a
were
analyzed
by LC/MS.
WesternPepMap™
blotting using
against
HSP90,
3 hr. 5-30%
gradient
(A: water,
formic
acid; B: vimentin
acetonitrile,
formic acid)
at
EGFR,
VDAC,
calreticulin,
SP1,0.1%
histone
3, HDAC2,
and0.1%
cytokeratin
18 was
300 nL/min
flow rate.
also
performed.
Overview
Introduction
Introduction
Methods
Methods
Mass Spectrometry
Liquid
Chromatography
Spectra were
using
an Orbitrap
Fusion
mass spectrometer
using topUltimate
speed
Samples
wereacquired
separated
by RP-HPLC
using
a Thermo
Scientific™ Dionex™
FTMSsystem
full scan
at 120,000
FT MS2
HCD 30KEASY-Spray™
resolution or ITcolumn,
MS2 CID
3000
connected
to aand
Thermo
Scientific™
15 and
cm ×FT75
1
MS3orHCD
(SPS Scientific™
using 10 fragments
from
the MS2 C18
spectra).
µm
a Thermo
Acclaim™
PepMap™
column, 50 cm × 75 µm over a
3 hr. 5-30% gradient (A: water, 0.1% formic acid; B: acetonitrile, 0.1% formic acid) at
Data Analysis
300 nL/min flow rate.
Spectral data files were analyzed using Thermo Scientific™ Proteome Discoverer™
Mass Spectrometry
1.4 software using the SEQUEST®HT search engine with a precursor mass tolerance
of 10 ppm
andacquired
fragmentusing
massantolerance
0.02 Da.
Carbamidomethylation
(+57.021
Spectra
were
Orbitrap of
Fusion
mass
spectrometer using top
speed
Da) forfull
cysteine
TMT isobaric
(+229.162
Da) foror
lysine
andCID
N-terminus
FTMS
scan atand
120,000
and FT labeling
MS2 HCD
30K resolution
IT MS2
and FT
1
residues
treated
modifications
oxidation
MS3
HCDwere
(SPS
using as
10 static
fragments
from the with
MS2variable
spectra).methionine
(+15.996 Da). Data was searched against a Swiss-Prot® human database with a 1%
Data Analysis
FDR criteria using Percolator.
Spectral data files were analyzed using Thermo Scientific™ Proteome Discoverer™
Proteome Discoverer 1.4 software was used to calculate TMT reporter ratios with mass
1.4 software using the SEQUEST®HT search engine with a precursor mass tolerance
tolerance ±10 ppm without applying the isotopic correction factors. A protein ratio was
of 10 ppm and fragment mass tolerance of 0.02 Da. Carbamidomethylation (+57.021
expressed as a median value of the ratios for all quantifiable spectra of the peptides
Da) for cysteine and TMT isobaric labeling (+229.162 Da) for lysine and N-terminus
pertaining to that protein.
residues were treated as static modifications with variable methionine oxidation
(+15.996 Da). Data was searched against a Swiss-Prot® human database with a 1%
FDR criteria using Percolator.
expressed asFractionation
a median value
the ratios for
all quantifiable
spectra of the peptides
Subcellular
byofDifferential
Detergent
Extraction
pertaining to that protein.
Separation of proteins by subcelluar localization is one method to enrich for proteins
while maintaining some biological context. Differential detergent extraction is a classic
biochemical technique used to separate proteins based on sequential solubilization of
cellular compartments using different detergents (Figure 1).2 As this fractionation
method does not require preparation of density gradients or ultracentrifugation,
fractionation is more easily accomplished using low speed microcentrifugation in less
Subcellular Fractionation by Differential Detergent Extraction
time with high reproducibility. However, the resolution of this technique is somewhat
limited to due
to the number
of fractions
collected,
of organelle
Separation
of proteins
by subcelluar
localization
is co-solubilization
one method to enrich
for proteins
compartments
andsome
poor biological
detergentcontext.
solubilityDifferential
of some protein
complexes.
while
maintaining
detergent
extraction is a classic
biochemical technique used to separate proteins based on sequential solubilization of
fractionation
cellular
compartmentsoverview
using different
(Figure
1).2 As this
Figure 1.Schematic
of thedetergents
subcellular
fractionation
procedure.
Cellular
method
does notare
require
preparation
of density
or ultracentrifugation,
compartments
sequentially
extracted
by gradients
incubating
cells with cytoplasmic
fractionation
is more
easily
accomplished
using lowextraction
speed microcentrifugation
in less
extraction buffer
(CEB)
followed
by membrane
buffer (MEB) and
nuclear
time
with high
reproducibility.
However,
the resolution
of this
technique
is somewhat
extraction
buffer
(NEB). Adding
micrococcal
nuclease
(MNase)
to NEB
extracts
limited
to due to theproteins
number of
fractions
collected,
co-solubilization
of pellet
organelle
chromatin-bound
from
the cell
pellet before
adding the
extraction
compartments
and
poor detergent
solubility
of some protein complexes.
buffer (PEB) to
solubilize
cytoskeletal
proteins.
Results
Ch
Us
we
pr
co
pr
an
Ch
en
wa
Us
we
Fi
pro
sp
co
pro
an
en
wa
Fig
sp
Figure 1.Schematic overview of the subcellular fractionation procedure. Cellular
compartments are sequentially extracted by incubating cells with cytoplasmic
extraction buffer (CEB) followed by membrane extraction buffer (MEB) and nuclear
extraction buffer (NEB). Adding micrococcal nuclease (MNase) to NEB extracts
chromatin-bound proteins from the cell pellet before adding the pellet extraction
buffer (PEB) to solubilize cytoskeletal proteins.
CEB
MEB
NEB
NEB
+MNase
PEB
FIGURE
2. Schematic
Reagent sampleNEB
preparation andPEB
LC-MS
CEB
MEBof TMT6plexNEB
analysis. A) Whole cell extracts and subcelluar protein
fractions from HeLa cell
+MNase
line are reduced, alkylated, and digested before being labeled with TMT6plex
reagents. Labeled peptides from each treatment condition are combined,
fractionated by high pH reverse phase chromatography and analyzed by LC/MS
analysis. B) TMT6plex-labeled peptides co-elute during MS acquisition but
generate unique reporter ion masses during MSn for relative quantitation.
FIGURE 2. Schematic of TMT6plex Reagent sample preparation and LC-MS
analysis.
A) Whole cell extracts and subcelluar protein fractions from HeLa cell
A
line are reduced, alkylated, and digested before being labeled with TMT6plex
reagents. Labeled peptides from each treatment condition are combined,
fractionated by high pH reverse phase chromatography and analyzed by LC/MS
analysis. B) TMT6plex-labeled peptides co-elute during MS acquisition but
generate unique reporter ion masses during MSn for relative quantitation.
A
Fi
co
fo
1.2
B
1.0
Fig
0.8
co
for
0.6
1.2
B
0.4
1.0
0.2
0.8
0.0
0.6
0.4
0.2
Nuclear
Soluble
FIGURE 2. Schematic of TMT6plex Reagent sample preparation and LC-MS
analysis. A) Whole cell extracts and subcelluar protein fractions from HeLa cell
line are reduced, alkylated, and digested before being labeled with TMT6plex
reagents. Labeled peptides from each treatment condition are combined,
fractionated by high pH reverse phase chromatography and analyzed by LC/MS
analysis. B) TMT6plex-labeled peptides co-elute during MS acquisition but
generate unique reporter ion masses during MSn for relative quantitation.
B
A
omic coverage of cultured cells
Barbara Kaboord
ME
NE
CB
PE
Fi
su
HDAC2
Figure 4. Analysis of protein fractions using label-free quantitation of
compartment-specific marker proteins. Normalized integrated average peak area
for each Chromatin
fraction is shown.
Histone 3
Bound
1.2
Cytoskeletal
Pellet
0.6
Cytokeratin 18
CE
ME
NE
CB
PE
18
Cytoplasmic Membrane Associated
Nuclear Soluble
1.0
Chromatin
Bound
Cytoskeletal Pellet
Characterization of Subcellular Protein Fractionations by Mass Spectrometry
Using the Pierce Subcellular Protein Fractionation Kit for Cultured Cells Kit, HeLa cells
were separated into five distinct protein fractions. Western blotting for various marker
proteins show excellent separation of marker proteins associated with different cellular
compartments (Figure 3). Notably, the membrane extract was found to contain
proteins associated with plasma membranes (EGFR), mitochondrial lumen (VDAC)
and endoplasmic reticulum lumen (Calreticulin). In addition, pellet fractions showed
enrichment for some cytoskeletal proteins (Vimentin and Cytokeratin 18), but tubulin
was observed most abundantly in the cytoplasmic fraction (data not shown).
In order to identify additional proteins which fractionate with known marker proteins,
subcellular
protein fractions were analyzed separately using label-free MS and also
0.6
labeled with TMT6plex tags for MS/MS quantitation (Figure 2). MS results from labelfree
0.4 quantitation (Figure 4) and TMT reagents (Figure 5) correlated well with Western
blot analysis. Overall, more than 40,000 unique peptides corresponding to 5,337
protein
groups were identified from the combined subcellular protein fractions which
0.2
was 2.5-fold greater than un-fractionated whole cell lysate (Figure 6).
Figure 3. Western blotting analysis of protein fractions using compartmentspecific antibody markers.
EGFR
Calreticulin
SP1
Histone 3 Vimentin Cytokeratin
FigureHSP90
5. Representative
TMT quantitation
of HDAC2
protein enrichment
of marker
18
proteins in different subcellular fractions relative to whole cell lysate.
0.8
0.0
Cytoplasmic Membrane Associated
Intensity (103)
250
HSP90
[(n
25
5
Characterization of Subcellular Protein Fractionations by Western Blot Analysis
Cytoplasmic
sc
30
co
ce
Figure 4. Analysis of protein fractions using label-free quantitation of
compartment-specific marker proteins. Normalized integrated average peak area
0.0
for each
fractionEGFR
is shown.
HSP90
Calreticulin
SP1
HDAC2
Histone 3 Vimentin Cytokeratin
Barbara Kaboord
35
ca
10
0.2
B
40
in
15
Vimentin
0.8
1.2
45
Ta
fra
20
1.0
0.4
omic coverage of cultured cells
Nuclear Soluble
HSP90
Chromatin
Bound
Cytoskeletal Pellet
Ta
in
ca
sc
[(n
fra
co
ce
GO
An
ca
su
pa
co
rev
2).
pro
Ta
fu
200
Characterization of Subcellular Protein Fractionations by Western Blot Analysis
Characterization of Subcellular Protein Fractionations by Mass Spectrometry
GO
Using the Pierce Subcellular Protein Fractionation Kit for Cultured Cells Kit, HeLa cells
EGFRmarker
were separated into five distinct protein fractions. Western blotting for various
proteins show excellent separation of marker proteins associated with different cellular
compartments (Figure 3). Notably, the membrane extract was found to contain
proteinsMembrane
associated with plasma membranes (EGFR), mitochondrial lumen (VDAC)
VDAC
and endoplasmic
reticulum lumen (Calreticulin). In addition, pellet fractions
showed
Associated
enrichment for some cytoskeletal proteins (Vimentin and Cytokeratin 18), but tubulin
was observed most abundantly in the cytoplasmic fraction (data not shown).
In order to identify additional proteins which fractionate with known marker proteins,
subcellular protein fractions were analyzed separately using label-free MS and also
Whole Cell
Membrane
Nuclear
Chromatin
Cytoskeletal
labeled with
TMT6plexCytoplasmic
tags for MS/MS
quantitation
(Figure 2).
MS results
from
Lysate
Associated
Soluble
Bound
Pellet labelfree quantitation (Figure 4) and TMT reagents (Figure 5) correlated well with Western
blot analysis. Overall, more than 40,000 unique peptides corresponding to 5,337
Histone 3
protein groups were identified from the combined subcellular protein fractions which
was 2.5-fold greater than un-fractionated whole cell lysate (Figure 6).
An
ca
su
pa
co
rev
2).
pro
Calreticulin
Figure 3. Western blotting analysis of protein fractions using compartmentspecific antibody markers.
Figure 5. Representative TMT quantitation of protein enrichment of marker
proteins inWhole
different
subcellular
fractions
relative
to whole
cell lysate.
Cell
Cytoplasmic
Membrane
Nuclear
Chromatin
Cytoskeletal
150
100
50
0
Intensity (103)
350
300
250
200
150
100
50
0
Lysate
Cytoplasmic
Nuclear
Soluble
r
HDAC2
EGFR
Chromatin
Membrane
Bound
Associated
Histone 3
VDAC
250
Intensity (103)
SP1
HSP90
Associated
Soluble
Bound
Ta
fu
Pellet
HSP90
200
Figure 6. Number of identified proteins and peptides from in-gel digests of
subcellular protein fractions and whole cell lysate.
150
100
45,000
40,000
50
0
35,000
30,000350
25,000
Intensity (103)
r
CE
300
Whole Cell
Lysate
Cytoplasmic
Membrane
Associated
Nuclear
Soluble
Chromatin
Bound
Cytoskeletal
Pellet
Nuclear
Soluble
Chromatin
Bound
Cytoskeletal
Pellet
Histone 3
250
20,000200
Vimentin
Calreticulin
Cytoskeletal
Pellet
Nuclear
Soluble
Cytokeratin 18
SP1
CE
ME
NE
CB
PE
HDAC2
Figure 4. Analysis of protein fractions using label-free quantitation of
compartment-specific marker proteins. Normalized integrated average peak area
for each fraction is shown.
1.2
Chromatin
Bound
Histone 3
1.0
0.8
0.6
0.4
0.2
Vimentin
Cytoskeletal
Pellet
Cytokeratin 18
CE
ME
NE
CB
5,000
0
1.2 Cytoplasmic Membrane Associated
Nuclear Soluble
Chromatin
Bound
0
Whole Cell
Lysate
Cytoskeletal Pellet
2 Protein Fractionation by Subcellular Location to Enhance Proteomic Coverage of
Cytoplasmic
Cytoplasmic Membrane
Associated
Membrane
Associated
Nuclear
Soluble
Chromatin Cytoskeletal Combined
Bound
Pellet
Fractions
Whole Cell
Lysate
Figure 6. Number of identified
proteins and
peptides
in-gel digests of
Proteins Identified
Unique
Peptidesfrom
Identified
subcellular protein fractions and whole cell lysate.
Table 1. GO annotation enrichment of subcellular protein fractions. Average
45,000
integrated peak area and standard deviation for each identified protein was
40,000
calculated for individual fractions compared to whole cell lysate. Standard
35,000
scores [(fraction area - average total area) / standard deviation] and fold-change
[(natural log (area for fraction / average total area)] were calculated for each
30,000
fraction. Proteins with a standard score > 0.6 and fold change > 0.5 were
25,000
considered enriched. The enriched proteins for each fraction was annotated for
20,000
cellular compartments using a Uniprot database and canonical GO Terms.
15,000
10,000
Fraction
# Proteins
Enriched
Cytoplasmic
1291
0
Cytoplasmic Membrane
Associated
Membrane
Associated
PE
Figure 4. Analysis of protein fractions using label-free quantitation of
compartment-specific
marker
proteins.
Normalized
integrated
average peak area
HSP90
EGFR
Calreticulin
SP1
HDAC2
Histone 3 Vimentin Cytokeratin
for each fraction is shown.
18
0.8
100
10,000 50
5,000
0.0
1.0
15,000150
2088
GO Term
GO:0005737
Proteins
Annotated
% GO Term
Match
1089
84.4
1374
65.8
Nuclear
Chromatin Cytoskeletal Combined
(cytoplasm)
Soluble
Bound
Pellet
Fractions
GO:0016020
(membrane)
Proteins Identified
Unique Peptides Identified
Whole Cell
Lysate
GO:0005634
Nuclear Soluble
882
718
81.4
(nucleus)
Table 1. GO annotation enrichment of
subcellular protein fractions. Average
integrated peak area and standardGO:0003682
deviation for each identified protein was
Chromatin
(chromatinto whole cell
38 lysate. Standard
11.6
calculated for individual329
fractions compared
Bound
scores [(fraction area - average total binding)
area) / standard deviation] and fold-change
Cytoskeletal
GO:0005856
[(natural
log (area for fraction / average
total area)] were calculated for each
257
62
24.1
fraction.Pellet
Proteins with a standard (cytoskeleton)
score > 0.6 and fold change > 0.5 were
considered enriched. The enriched proteins for each fraction was annotated for
cellularCells
compartments using a Uniprot database and canonical GO Terms.
Cultured
Fraction
# Proteins
Enriched
GO Term
Proteins
Annotated
% GO Term
Match
C
R
C
©2
Wa
Sw
info
pro
Associated
10,000
5,000
Soluble
Proteins Identified
0
Bound
Pellet
Fractions
Lysate
Unique Peptides Identified
Conclusions

Membrane
Nuclear
Chromatin Cytoskeletal
Combined
Whole Cell
Table 1.Cytoplasmic
GO annotation
enrichment
of subcellular
protein fractions.
Average
Associated
Soluble
Bound
Pellet
Fractions
Lysate
integrated peak area and standard deviation for each identified protein was
Proteins
Identified
Unique
Identified
calculated for individual
fractions
compared
toPeptides
whole cell
lysate. Standard
scores [(fraction area - average total area) / standard deviation] and fold-change
Table 1. GO
enrichment
of subcellular
fractions. for
Average
[(natural
logannotation
(area for fraction
/ average
total area)]protein
were calculated
each
integrated
peak area
standardscore
deviation
each
was
and
foldidentified
change >protein
0.5 were
fraction.
Proteins
withand
a standard
> 0.6for
calculated for
individual
to whole
cell lysate.
considered
enriched.
Thefractions
enrichedcompared
proteins for
each fraction
wasStandard
annotated for
scores [(fraction
area - using
average
total area)
/ standard
deviation] GO
andTerms.
fold-change
cellular
compartments
a Uniprot
database
and canonical
[(natural log (area for fraction / average total area)] were calculated for each
# Proteins
Proteins
% GO
Term
fraction.
Proteins with
a standard score
> 0.6 and fold
change > 0.5
were
Fraction
GO Term
Annotated
Match
considered enriched.Enriched
The enriched proteins for each
fraction was annotated
for
GO:0005737
cellular
compartments 1291
using a Uniprot database and 1089
canonical GO Terms.
Cytoplasmic
84.4
(cytoplasm)
# Proteins
Proteins
% GO Term
Membrane
GO:0016020
Fraction
GO Term
2088
1374
65.8
Enriched
Annotated
Match
Associated
(membrane)
GO:0005737
GO:0005634
Cytoplasmic
1291
1089
84.4
882
718
81.4
Nuclear
Soluble
(cytoplasm)
(nucleus)
Membrane
GO:0016020
GO:0003682
2088
1374
65.8
Chromatin
Associated
(membrane)
329
(chromatin
38
11.6
Bound
GO:0005634
binding)
Nuclear Soluble
882
718
81.4
(nucleus)
Cytoskeletal
GO:0005856
257
62
24.1
GO:0003682
Pellet
(cytoskeleton)
Chromatin
329
(chromatin
38
11.6
Bound
binding)
Cytoskeletal
GO:0005856
GO Analysis
of Subcellular
257 Protein Fractionations
62
24.1
Pellet
(cytoskeleton)
Annotation of proteins by cellular compartment revealed significant enrichment for
canonical GO terms (Table 1). However, as most proteins are annotated for multiple
subcellular locations, some terms did not show as much enrichment as others. In
particular, proteins found in the chromatin bound and cytoskeletal pellet fractions
correlated less with the subcelluar GO terms. Further analysis of proteins by function
revealed significant increases in additional protein groups for different fractions (Table
2). Notably, the membrane associated fraction showed enrichment for mitochondrial
proteins and the pellet fraction showed enrichment of lipid raft associated proteins.
mitochondrial inner membrane
4.7
presequence translocase complex
heterotrimeric
G-protein
complex
Separation of proteins by subcelluar
localization
reduces
sample complexity4.5
and
increases protein identifications while maintaining biological context.
Conclusions
 Reproducible fractionation at the protein and peptide level enabled deeper





proteomic coverage and more precise assignment of protein localization.
Separation of proteins by subcelluar localization reduces sample complexity and
increases
protein identifications
while
biological well
context.
Protein
expression
ratios from TMT
ionmaintaining
reporters correlated
with label-free
and Western blot analysis.
Reproducible fractionation at the protein and peptide level enabled deeper
proteomic
coverage
andshowed
more precise
assignment
of protein
localization.
GO
annotation
analysis
significant
enrichment
for identified
proteins in
each fraction and assignment of additional protein functional groups to
Protein expression ratios from TMT ion reporters correlated well with label-free
subcellular protein fractions based on detergent solubility.
and Western blot analysis.
 GO annotation analysis showed significant enrichment for identified proteins in
References
each fraction and assignment of additional protein functional groups to
subcellular G.C.,
protein
on detergent
solubility.
1. McAllister,
et fractions
al. 2014. based
MultiNotch
MS3 enables
accurate, sensitive, and
multiplexed detection of differential expression across cancer cell line proteomes.
Anal Chem 86(14):7150-8.
References
2. Walker, JM. The Proteomics Handbook. Humana Press. 2005. Differential
1. McAllister, G.C., et al. 2014. MultiNotch MS3 enables accurate, sensitive, and
detergent fractionation of eukaryotic cells. p37-48.
multiplexed detection of differential expression across cancer cell line proteomes.
Anal Chem
86(14):7150-8.
© 2015 Thermo
Fisher Scientific
Inc. All rights reserved. SEQUEST is a registered trademark of the University of
Washington. Swiss-Prot is a registered trademark of Institut Suisse de Bioinformatique (Sib) Foundation
2. Walker,
JM.
The Proteomics
Handbook.
Press.
2005.
Differential
Switzerland.
All other
trademarks
are the property
of Thermo Humana
Fisher Scientific
Inc. and
its subsidiaries.
This
information
is not intended
to encourage
of these products
any manners that might infringe the intellectual
detergent
fractionation
ofuse
eukaryotic
cells. inp37-48.
property rights of others.
© 2015 Thermo Fisher Scientific Inc. All rights reserved. SEQUEST is a registered trademark of the University of
Washington. Swiss-Prot is a registered trademark of Institut Suisse de Bioinformatique (Sib) Foundation
Switzerland. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This
information is not intended to encourage use of these products in any manners that might infringe the intellectual
property rights of others.
Table 2. Identification of additional enriched GO annotations and protein
function enriched in subcellular protein fractions.
Fraction
GO Term
Fold Change
COP9 signalosome
Cytoplasmic
Membrane Associated
Nuclear Soluble
Chromatin Bound
Cytoskeletal Pellet
BRISC complex
SNARE complex
BRCA1-A complex
prefoldin complex
mitochondrial ribosome
mitochondrial respiratory chain complex I
SNARE complex
mitochondrial small ribosomal subunit
mitochondrial large ribosomal subunit
mitochondrial respiratory chain complex I
histone methyltransferase complex
core TFIIH complex
transcription factor TFIID complex
transcriptional repressor complex
Smc5-Smc6 complex
chromocenter
MIS12/MIND type complex
U2 snRNP
condensed nuclear chromosome,
centromeric region
chromocenter
Ragulator complex
vacuolar proton-transporting V-type
ATPase complex
mitochondrial inner membrane
presequence translocase complex
heterotrimeric G-protein complex
4.1
3.2
3.2
3.2
3.2
7.8
6.2
5.9
5.4
4.9
5.1
4.3
3.6
3.6
3.3
7.1
7.1
6
4.8
4.8
6.9
4.7
4.7
4.7
4.5
Conclusions

Separation of proteins by subcelluar localization reduces sample complexity and
increases protein identifications while maintaining biological context.

Reproducible fractionation at the protein and peptide level enabled deeper
proteomic coverage and more precise assignment of protein localization.

Protein expression ratios from TMT ion reporters correlated well with label-free
and Western blot analysis.

GO annotation analysis showed significant enrichment for identified proteins in
each fraction and assignment of additional protein functional groups to
www.thermofisher.com
subcellular protein fractions based on detergent solubility.
©2016 Thermo Fisher Scientific Inc. All rights reserved.SEQUEST is a registered trademark of the University of Washington. Swiss-Prot is
a registered trademark of Institut Suisse de Bioinformatique (Sib) Foundation Switzerland. All other trademarks are the property of Thermo
Fisher Scientific and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific products.
It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.
Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales
1. McAllister, G.C., et al. 2014. MultiNotch MS3 enables accurate, sensitive, and
representative for details.
References
multiplexed detection of differential expression across cancer cell line proteomes.
86(14):7150-8.
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The4300
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