FI
Purpose: To demonstrate the applications of the new Thermo ScientificTM PierceTM
High pH Reversed Phase Peptide Fractionation Kit, together with Thermo ScientificTM
PierceTM Quantitative Colormetric and Fluorometric Peptide Assay kits in mass
spectrometry-based large-scale proteomic experiments to increase protein/peptide
identification numbers and protein sequence coverage with high analytical
reproducibility.
Methods: HeLa lysate digests, both native and Thermo ScientificTM Tandem Mass
TagTM (TMT) reagents, were fractionated off-line in a high pH reversed-phase mode
using spin-columns packed with polymer-based hydrophobic resin and bench top
microcentrifuge, and individual fractions were analyzed by LC-MS. Absolute peptide
quantities were determined prior to fractionation and sample injection using the
quantitative peptide assays.
Th
0.
co
pe
bo
ac
Results: Protein IDs increased by ~1.8 times and ~3 times for native and TMT-labeled
samples, respectively, with fractionation of the complex digest samples.
1
Sergei I. Snovida1; Xiaoyue Jiang2; Ramesh Ganapathy1; Sijian
; Ryanrelevant
Bomgarden1;
ManyHou
biologically
changes in the proteome occur at the mid-to-low range of
the protein abundance scale. Off-line fractionation of complex peptide mixtures from
Paul Haney1; Rosa Viner2; John C. Rogers1
sample digests enables deeper proteome sequencing through increased protein
1
Thermo Fisher Scientific, Rockford, IL, USA; 2Thermo Fisher
Scientific, San Jose, CA, USA
Poster Note 6445 7
High pH Reversed-Phase
Peptide Fractionation in a
Convenient Spin-Column Format
High pH Reversed-Phase PeptideIntroduction
Fractionation in a Convenient Sp
identifications and sequence 1
coverage. Similar to strong cation
exchange (SCX)
1; Ryan
Sergei I. Snovida1; Xiaoyue Jiang2; Ramesh
Ganapathy
; Sijian
Houfractionation
peptide
fractionation methods, high
pH reversed-phase
enablesBomgarde
orthogonal peptide fractionation to low pH reversed-phased separation. In contrast to
1Thermo Fisher Scientific, Rockford, IL, USA;
2Thermo
SCX fractionation,
samples fractionated
by high
pH reversed-phase fractionation
do
Fisher
Scientific,
San Jose,
CA
not require desalting before LC-MS analysis. In this study, we assessed peptide/protein
identification numbers, fractional resolution, and reproducibility of high pH reversedphase fractionation in a convenient, spin column format.
Methods
Purpose: To demonstrate the applications of the new Thermo ScientificTM PierceTM
High pH Reversed Phase Peptide Fractionation Kit, together with Thermo ScientificTM
PierceTM Quantitative Colormetric and Fluorometric Peptide Assay kits in mass
spectrometry-based large-scale proteomic experiments to increase protein/peptide
identification numbers and protein sequence coverage with high analytical
reproducibility.
Methods: HeLa lysate digests, both native and Thermo
Mass
TagTM (TMT) reagents, were fractionated off-line in a high pH reversed-phase mode
using spin-columns packed with polymer-based hydrophobic resin and bench top
microcentrifuge, and individual fractions were analyzed by LC-MS. Absolute peptide
quantities were determined prior to fractionation and sample injection using the
quantitative peptide assays.
ScientificTM Tandem
Results: Protein IDs increased by ~1.8 times and ~3 times for native and TMT-labeled
samples, respectively, with fractionation of the complex digest samples.
Introduction
F
Protein
extracts
from
HeLa lysates
were digested
sequentially
with Lys-C
trypsin.
The
column
is first
conditioned
by successive
washes
with acetonitrile
andand
aqueous
Initial TFA
protein
concentrations
were
determined
a BCA
assay.
Subsequent
0.1%
by spinning
at 5,000
g (A);
a sampleusing
solution
is then
loaded
onto the peptide
quantitation
wasflowthrough
performed using
a Pierce
Peptide
Quantitation
Assay
column
and the
is collected
by Colorimetric
centrifugation
at 3,000
g (B); bound
(Product#are
23275)
and/or
Pierce
Fluorometric
Peptide
Quantitation
Assay
(Product#
peptides
further
washed
to remove
any salts
and/or
weakly bound
chemicals
(C);
23290).sample
Portions
of the digested
samples were
with
Tandem
Tag (TMT)
bound
peptides
are then fractionated
bylabeled
stepwise
elution
withMass
increasing
reagents. Pierce
High pH solutions
Reversed-Phase
Fractionation
Kits (Product# 84868) were
acetonitrile
concentration
at high pH
(D).
used to fractionate both native and TMT-labeled digest samples into eight fractions by
an increasing acetonitrile step-gradient elution. Fractions were dried in a vacuum
centrifuge and re-suspended in 0.1% formic acid prior to LC-MS analysis.
T
(H
sa
A
Liquid
(1) Acetonitrile (x2)
Chromatography
(2) 0.1% TFA (x2)
Methods
Conditioned
spin-column
Digest
TM
ScientificTM Dionex
sample
Liquid chromatography was performed using Thermo
UltimateTM
3000 Nano LC system, utilizing a 50 cm C18 Thermo ScientificTM EASY-SprayTM column
heated at 60°C. Two- or three-hour gradients were used in all experiments.
Centrifuge at 3,000g
Mass Spectrometry
Many biologically relevant changes in the proteome occur at the mid-to-low range of
the protein abundance scale. Off-line fractionation of complex peptide mixtures from
sample digests enables deeper proteome sequencing through increased protein
identifications and sequence coverage. Similar to strong cation exchange (SCX)
peptide fractionation methods, high pH reversed-phase fractionation enables
orthogonal peptide fractionation to low pH reversed-phased separation. In contrast to
SCX fractionation, samples fractionated by high pH reversed-phase fractionation do
not require desalting before LC-MS analysis. In this study, we assessed peptide/protein
identification numbers, fractional resolution, and reproducibility of high pH reversedphase fractionation in a convenient, spin column format.
Centrifuge at 5,000g
(2 minutes)
(2 minutes)
All samples were analyzed on a Thermo ScientificTM Orbitrap FusionTM TribridTM mass
spectrometer.
Flowthrough
B
For native (unlabeled) samples, MS-level scans fraction
were performed with Orbitrap
resolution set to 60,000; AGC Target 2.0e5; maximum injection time 50 ms; intensity
Waste
threshold 5.0e3; dynamic exclusion 45 sec. Data dependent MS2 selection was
performed in Top Speed mode with HCD collision energy set to 28% and ion trap
detection (AGC target 1.0e4, maximum injection time 35 ms).
Water
For TMT-labeled samples, MS-level scans were performed with Orbitrap resolution
set
D
to 120,000; Elution
AGCsolution
target
4.0e5; maximum injection time 50 ms; intensity threshold
1
2
5.0e3; dynamic exclusion 60 sec. Data-dependent MS selection was performed in Top
Speed mode with CID collision energy set to 35%. Data-dependent MS3 was
performed in Top N mode with Synchronous Precursor Selection set to 10Centrifuge
precursors
at 3,000g
at 3,000g
and HCDCentrifuge
collision
energy set to 65% and Orbitrap resolution set to 60,000. (2 minutes)
(2 minutes)
Protein extracts from HeLa lysates were digested sequentially with Lys-C and trypsin.
Initial protein concentrations were determined using a BCA assay. Subsequent peptide
quantitation was performed using a Pierce Colorimetric Peptide Quantitation Assay
(Product# 23275) and/or Pierce Fluorometric Peptide Quantitation Assay (Product#
23290). Portions of the digested samples were labeled with Tandem Mass Tag (TMT)
reagents. Pierce High pH Reversed-Phase Fractionation Kits (Product# 84868) were
used to fractionate both native and TMT-labeled digest samples into eight fractions by
an increasing acetonitrile step-gradient elution. Fractions were dried in a vacuum
centrifuge and re-suspended in 0.1% formic acid prior to LC-MS analysis.
Liquid Chromatography
Liquid chromatography was performed using Thermo ScientificTM DionexTM UltimateTM
3000 Nano LC system, utilizing a 50 cm C18 Thermo ScientificTM EASY-SprayTM column
heated at 60°C. Two- or three-hour gradients were used in all experiments.
Mass Spectrometry
ScientificTM
Orbitrap
FusionTM
TribridTM
mass
For native (unlabeled) samples, MS-level scans were performed with Orbitrap
resolution set to 60,000; AGC Target 2.0e5; maximum injection time 50 ms; intensity
threshold 5.0e3; dynamic exclusion 45 sec. Data dependent MS2 selection was
performed in Top Speed mode with HCD collision energy set to 28% and ion trap
detection (AGC target 1.0e4, maximum injection time 35 ms).
For TMT-labeled samples, MS-level scans were performed with Orbitrap resolution set
to 120,000; AGC target 4.0e5; maximum injection time 50 ms; intensity threshold
5.0e3; dynamic exclusion 60 sec. Data-dependent MS2 selection was performed in Top
Speed mode with CID collision energy set to 35%. Data-dependent MS3 was
performed in Top N mode with Synchronous Precursor Selection set to 10 precursors
and HCD collision energy set to 65% and Orbitrap resolution set to 60,000.
Data Analysis
All .raw files were processed using Thermo ScientificTM Proteome DiscovererTM 1.4
software. Data was searched against a custom human/yeast database using Sequest®
HT search engine.
Data Analysis
All .raw files were processed using Thermo ScientificTM Proteome
DiscovererTM 1.4C
Wash
software. Data was searched against a custom human/yeast
database using Sequest®
fraction
HT search engine.
Peptide elution with increasing acetonitrile step-gradient
1
2
3
4
5
6
7
RT: 0.00
100
80
60
40
20
R
0
100
Hi
80
60
To
we
20
pH
so
0
100
Th
80
ad
60
co
pr
40
40
20
0
Re
0
An
co
sim
FI
pr
id
Ba
la
re
in
RT: 0.00
100
80
R
60
8
40
Sample fractions
20
0
100
Results
High pH reversed-phase fractionation orthogonality
Relative Abundance
Sample Preparation
All samples were analyzed on a Thermo
spectrometer.
Sample Preparation
FIGURE
1. Off-line spin-column high pH reversed-phase fractionation workflow.
Relative Abundance
Overview
To demonstrate orthogonality of low and high pH reversed phase separation modes,
we fractionated 100 µg of HeLa digest according to the workflow in Figure 1 using low
pH (0.1% trifluoroacetic acid, TFA) and high pH (0.1% triethylamine, TEA) elution
solutions at 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, and 50% acetonitrile steps.
The fractions were then analyzed individually by LC-MS (with 0.1% formic acid, FA,
additive in the mobile phases). As shown in Figure 2, peptides in the high pH fractions
cover more chromatographic space due to changes in hydrophobic retention
properties during fractionation at high pH.
80
R
60
40
20
0
100
80
R
60
40
20
0
0
Reproducibility and fractional resolution
Im
Analysis of the replicate sets of fractions revealed excellent reproducibility of the spincolumn fractionation format. Fractional base peak chromatogram profiles shared high
similarity across different replicate fractions (Figure 3). Reproducibility of the elution
profiles, in terms of unique peptides identified and the numbers of unique peptides
identified, was also good among the replicates for both the native (Figure 4) and TMTlabeled samples.
Du
ph
Th
pr
He
gr
fra
sa
FIGURE
FIGURE1.1.Off-line
Off-linespin-column
spin-columnhigh
highpH
pHreversed-phase
reversed-phasefractionation
fractionationworkflow.
workflow.
FIGURE
FIGURE2.2.Orthogonality
Orthogonalityof
oflow
lowand
andhigh
highpH
pHreversed-phase
reversed-phasefractionation.
fractionation.
The
Thecolumn
columnisisfirst
firstconditioned
conditionedby
bysuccessive
successivewashes
washeswith
withacetonitrile
acetonitrileand
andaqueous
aqueous
0.1%
0.1%TFA
TFAby
byspinning
spinningatat5,000
5,000gg(A);
(A);aasample
samplesolution
solutionisisthen
thenloaded
loadedonto
ontothe
the
column
columnand
andthe
theflowthrough
flowthroughisiscollected
collectedby
bycentrifugation
centrifugationatat3,000
3,000gg(B);
(B);bound
bound
peptides
peptidesare
arefurther
furtherwashed
washedtotoremove
removeany
anysalts
saltsand/or
and/orweakly
weaklybound
boundchemicals
chemicals(C);
(C);
bound
boundsample
samplepeptides
peptidesare
arethen
thenfractionated
fractionatedby
bystepwise
stepwiseelution
elutionwith
withincreasing
increasing
acetonitrile
acetonitrileconcentration
concentrationsolutions
solutionsatathigh
highpH
pH(D).
(D).
TIC
TICchromatograms
chromatogramsofoffractions
fractionseluted
elutedwith
with7.5%
7.5%acetonitrile
acetonitrilesolution
solutionatathigh
highpH
pH
(High-low)
(High-low)and
andlow
lowpH
pH(Low-low)
(Low-low)are
arecompared
comparedtotothat
thatofofunfractionated
unfractionateddigest
digest
sample.
sample.
100
100
8080
6060
High→low
High→low
pH
pH
(1)
(1)Acetonitrile
Acetonitrile(x2)
(x2)
(2)
(2)0.1%
0.1%TFA
TFA(x2)
(x2)
2020
Conditioned
Conditioned
spin-column
spin-column
00
100
100
Digest
Digest
sample
sample
Centrifuge
Centrifugeatat3,000g
3,000g
(2(2minutes)
minutes)
Centrifuge
Centrifugeatat5,000g
5,000g
(2(2minutes)
minutes)
Relative Abundance
Relative Abundance
AA
6060
2020
Water
Water
Centrifuge
Centrifugeatat3,000g
3,000g
(2(2minutes)
minutes)
Centrifuge
Centrifugeatat3,000g
3,000g
(2(2minutes)
minutes)
Peptide
Peptideelution
elutionwith
withincreasing
increasingacetonitrile
acetonitrilestep-gradient
step-gradient
Wash
Wash
fraction
fraction
CC
22
33
44
55
66
77
53.43
53.43
3030
4040
55.54
55.54
unfractionated
unfractionated
94.89
94.89
88.16
88.16
5050
6060
7070
8080
Time
(min)
Time
(min)
NL:
NL:
6.74E9
6.74E9
TIC
TICMS
MS
hela_td_2u
hela_td_2u
gload_test
gload_test
104.15
104.15
95.40
95.40
9090
117.08
110.92
110.92117.08
118.98
118.98
100
100
110
110
120
120
134.96
134.96
144.14
144.14
137.44
137.44
130
130
140
140
157.34
157.34
B.
B.U
150
150
Fraction
Fraction12.5%ACN
12.5%ACN
63.80
63.80
100
100
73.04
73.04
69.84
69.84
56.37
54.37
54.37 56.37
82.77
82.77
53.44
53.44
4.57
4.57
36.91
36.91
31.48
31.48
26.73
21.73
21.73 26.73
94.09
94.09 103.29
103.29
115.96
115.96
123.01
123.01
135.45
135.45
132.07
132.07
138.03
138.03
63.98
63.98
Replicate
Replicate22
8080
54.30
54.30
58.28
58.28
6060
4040
2020
00
100
100
38.50
38.50
54.56
54.56
44.73
44.73
38.40
38.40
93.51
93.51
135.43
135.43
115.77123.21
94.19
94.19 111.20
123.21
111.20115.77
137.88
131.88
131.88 137.88
144.02
144.02
70.36
70.3673.06
73.06
55.16
55.16
94.46
94.46
101.54
101.54 111.78
111.78
6060
NL:
NL:
9.35E8
9.35E8
Base
Base
Peak
PeakMS
MS
sepax2_3_15mg_h
sepax2_3_15mg_h
ela_high_ph_rp_2ug
ela_high_ph_rp_2ug
_total_load_per_set
_total_load_per_set
_c_12_5%acn
_c_12_5%acn
82.91
82.91
73.85
73.85
93.75
93.75
85.04
85.04
2020
5050
159.67
159.67
NL:
NL:
1.01E9
1.01E9
Base
Base
Peak
PeakMS
MS
sepax2_3_15mg_h
sepax2_3_15mg_h
ela_high_ph_rp_2ug
ela_high_ph_rp_2ug
_total_load_per_set
_total_load_per_set
_b_12_5%acn
_b_12_5%acn
92.04
92.04
64.25
64.25
6060
35.97
35.97
31.70
31.70
28.04
28.04
24.79
7.14
7.1410.86
10.86 24.79
00
00
1010
2020
3030
4040
82.85
82.85
78.16
78.16
36.80
31.86
31.8636.80
27.12
1.21
1.2111.44
11.4422.55
22.5527.12
8080
4040
69.97
69.97 73.04
73.04
53.28
53.28
45.95
45.95
Replicate
Replicate33
NL:
NL:
7.89E8
7.89E8
Base
Base
Peak
PeakMS
MS
sepax2_3_15mg_H
sepax2_3_15mg_H
eLa_high_pH_RP_2
eLa_high_pH_RP_2
ug_total_load_per_s
ug_total_load_per_s
et_A_12_5%ACN
et_A_12_5%ACN
84.51
84.5191.94
91.94
93.54
93.54
46.18
38.59
38.59 46.18
2020
To
Todemonstrate
demonstrateorthogonality
orthogonalityofoflow
lowand
andhigh
highpH
pHreversed
reversedphase
phaseseparation
separationmodes,
modes,
we
wefractionated
fractionated100
100µg
µgofofHeLa
HeLadigest
digestaccording
accordingtotothe
theworkflow
workflowininFigure
Figure11using
usinglow
low
pH
pH(0.1%
(0.1%trifluoroacetic
trifluoroaceticacid,
acid,TFA)
TFA)and
andhigh
highpH
pH(0.1%
(0.1%triethylamine,
triethylamine,TEA)
TEA)elution
elution
solutions
solutionsatat5%,
5%,7.5%,
7.5%,10%,
10%,12.5%,
12.5%,15%,
15%,17.5%,
17.5%,20%,
20%,and
and50%
50%acetonitrile
acetonitrilesteps.
steps.
The
Thefractions
fractionswere
werethen
thenanalyzed
analyzedindividually
individuallyby
byLC-MS
LC-MS(with
(with0.1%
0.1%formic
formicacid,
acid,FA,
FA,
additive
additiveininthe
themobile
mobilephases).
phases).As
Asshown
shownininFigure
Figure2,2,peptides
peptidesininthe
thehigh
highpH
pHfractions
fractions
cover
covermore
morechromatographic
chromatographicspace
spacedue
duetotochanges
changesininhydrophobic
hydrophobicretention
retention
properties
propertiesduring
duringfractionation
fractionationatathigh
highpH.
pH.
80.41
80.41
81.05
78.59
78.59 81.05
RT:
RT:0.00
0.00
- 160.00
- 160.00
4040
Relative Abundance
Relative Abundance
High
HighpH
pHreversed-phase
reversed-phasefractionation
fractionationorthogonality
orthogonality
64.56
64.56
145.12146.94
137.12145.12
123.31
123.31128.38
128.38 137.12
146.94
106.11
106.11
Base
Basepeak
peakchromatograms
chromatogramsofofthree
threereplicate
replicatefractions
fractionsare
arecompared.
compared.Good
Good
reproducibility
reproducibilityamong
amongreplicate
replicatefraction
fractionsets
setsisisevidenced
evidencedby
by high
highdegree
degreeofofsimilarity
similarity
ininthe
thebase
basepeak
peakchromatogram
chromatogramprofiles.
profiles.
00
100
100
Results
Results
67.59
67.5972.56
72.56 82.95
82.9588.04
88.0496.29
96.29
70.64
70.64
FIGURE
FIGURE3.3.Fractionation
Fractionationreproducibility
reproducibilitywith
withspin-columns.
spin-columns.
6060
Sample
Samplefractions
fractions
A.
A.U
NL:
NL:
2.82E9
2.82E9
TIC
TICMS
MS
hela_100ug
hela_100ug
_lowph_col
_lowph_col
umn_7_5%
umn_7_5%
acn
acn
51.19
51.19
Replicate
Replicate11
88
NL:
NL:
2.37E9
2.37E9
TIC
TICMS
MS
HeLa_100u
HeLa_100u
g_50C_8w
g_50C_8w
eeks_colum
eeks_colum
n_7_5%AC
n_7_5%AC
NN
Nu
N
ar
a
on
o
Ne
N
ide
id
sa
s
48.59
48.59
30.13
30.13
27.51
27.51
22.96
15.81
15.8122.96
2020
137.21
137.21
145.08
145.08150.24
134.38
134.38
150.24
44.85
44.85
39.39
39.39 47.29
47.29
37.61
37.61
36.62
36.62
35.40
35.40
33.89
33.89
32.95
32.95
0.45
0.45
00
00
1010
116.45
116.45
7.5%ACN
7.5%ACNlow
lowpH
pH
40.75
40.75
44.17
44.17
8080
11
103.42
103.42
39.36
36.71
36.7139.36
30.16
30.16
29.38
29.38
27.70
27.70
23.19
6.45
6.4514.06
14.0623.19
2020
DD
101.31
101.31
87.64
87.64
32.91
32.91
32.16
32.16
4040
Elution
Elutionsolution
solution1 1
39.78
39.78
94.38
94.38
35.99
35.99
34.68
34.68
6060
7.5%ACN
7.5%ACNhigh
highpH
pH
62.38
62.38
60.06
60.06
72.92
64.72
64.7272.92
66.14
66.14
78.61
78.61
83.57
83.57
118.23
118.23
Low→low
Low→low
pH
pH
8080
Waste
Waste
47.23
47.23
28.33
28.33
25.35
25.35
20.47
5.84
5.8412.16
12.1620.47
4040
00
100
100
BB
Flowthrough
Flowthrough
fraction
fraction
8080
51.28
51.28
39.48
39.48
34.69
34.69
33.66
33.66
32.69
32.69
30.01
30.01
4040
n
n
RT:
RT:0.00
0.00
- 160.00
- 160.00
FI
F
na
n
7070
8080
9090
Time
(min)
Time
(min)
100
100
110
110
123.63
123.63
137.25144.08
135.03
135.03137.25
144.08 158.71
158.71
120
120
130
130
140
140
150
150
Reproducibility
Reproducibilityand
andfractional
fractionalresolution
resolution
Improvement
Improvementin
inprotein
proteinidentification
identificationnumbers
numbers
Analysis
Analysisofofthe
thereplicate
replicatesets
setsofoffractions
fractionsrevealed
revealedexcellent
excellentreproducibility
reproducibilityofofthe
thespinspincolumn
columnfractionation
fractionationformat.
format.Fractional
Fractionalbase
basepeak
peakchromatogram
chromatogramprofiles
profilesshared
sharedhigh
high
similarity
similarityacross
acrossdifferent
differentreplicate
replicatefractions
fractions(Figure
(Figure3).
3).Reproducibility
Reproducibilityofofthe
theelution
elution
profiles,
profiles,ininterms
termsofofunique
uniquepeptides
peptidesidentified
identifiedand
andthe
thenumbers
numbersofofunique
uniquepeptides
peptides
identified,
identified,was
wasalso
alsogood
goodamong
amongthe
thereplicates
replicatesfor
forboth
boththe
thenative
native(Figure
(Figure4)
4)and
andTMTTMTlabeled
labeledsamples.
samples.
Due
Duetotothe
theoverall
overalldecrease
decreaseininchromatographic
chromatographicdensity
densityinineach
eachhigh
highpH
pHreversed
reversed
phase
phasefraction,
fraction,we
weare
areable
abletotosample
samplemore
morepeptides
peptidesand
andacquire
acquiremore
moreMS
MS22spectra.
spectra.
This
Thisleads
leadstotomore
morespectral
spectralmatches,
matches,more
moreunique
uniquepeptide
peptideidentifications,
identifications,and
andmore
more
protein
proteinidentifications
identificationswith
withhigher
highersequence
sequencecoverage.
coverage.Upon
Uponfractionation
fractionationofofnative
native
HeLa
HeLadigests
digestsand
andanalysis
analysisofofthe
theindividual
individualfractions,
fractions,nearly
nearly1.8
1.8times
timesmore
moreprotein
protein
groups
groupswere
wereidentified
identified(Figure
(Figure3).
3).Similar
Similaranalysis
analysisofofthe
theTMT-labeled
TMT-labeledHeLa
HeLasample
sample
fractions
fractionsrevealed
revealednearly
nearly33times
timesmore
moreprotein
proteingroups
groupscompared
comparedtotothe
theunfractionated
unfractionated
samples.
samples.
2 High pH Reversed-Phase Peptide Fractionation in a Convenient Spin-Column Format
D.
D.OO
idid
9
MS
0u
_100u
C_8w
w
m
colum
C
%AC
FIGURE
FIGURE
4. 4.
Reproducibility
Reproducibility
ofof
fractional
fractional
profiles
profiles
and
and
fractional
fractional
resolution
resolution
ofof
native
native
digest
digest
samples.
samples.
FIGURE
FIGURE
5. 5.
Reproducibility
Reproducibility
ofof
fractional
fractional
profiles
profiles
and
and
fractional
fractional
resolution
resolution
ofof
TMT-labeled
TMT-labeled
digest
digest
samples.
samples.
Numbers
Numbers
of of
unique
unique
peptides
peptides
(A)(A)
and
and
protein
protein
groups
groups
(B)(B)
identified
identified
in in
a given
a given
fraction
fraction
are
are
similar
similar
across
across
the
the
triplicate
triplicate
set.
set.
Over
Over
70%
70%
of of
allall
peptides
peptides
were
were
identified
identified
in in
only
only
one
one
fraction,
fraction,
indicating
indicating
excellent
excellent
fractional
fractional
resolution
resolution
of of
the
the
spin-column
spin-column
format
format
(C).
(C).
Nearly
Nearly
2.5
2.5
times
times
more
more
unique
unique
peptides
peptides
and
and
1.8
1.8
times
times
more
more
protein
protein
groups
groups
were
were
identified
identified
upon
upon
the
the
analysis
analysis
of of
fractionated
fractionated
sample
sample
compared
compared
to to
unfractionated
unfractionated
samples
samples
(black
(black
bars).
bars).
Numbers
Numbers
of of
unique
unique
peptides
peptides
(A)(A)
and
and
protein
protein
groups
groups
(B)(B)
identified
identified
in in
a given
a given
fraction
fraction
are
are
similar
similar
across
across
the
the
triplicate
triplicate
set.
set.
Over
Over
75%
75%
of of
allall
peptides
peptides
were
were
identified
identified
in in
only
only
one
one
fraction,
fraction,
indicating
indicating
excellent
excellent
fractional
fractional
resolution
resolution
of of
the
the
spin-column
spin-column
format
format
(C).
(C).
Over
Over
5 times
5 times
more
more
unique
unique
peptides
peptides
and
and
nearly
nearly
3 times
3 times
more
more
protein
protein
groups
groups
were
were
identified
identified
upon
upon
the
the
analysis
analysis
of of
fractionated
fractionated
sample
sample
compared
compared
to to
unfractionated
unfractionated
samples
samples
(black
(black
bars).
bars).
53815
53815
A.A.Unique
UniqueNative
NativePeptide
PeptideIDs
IDs
9
MS
ug
100ug
ol
h_col
%
7_5%
36960
36960
A.A.Unique
UniqueTMT-labeled
TMT-labeledPeptide
PeptideIDs
IDs
21911
21911
117
117
5050
5269
5269
1407711931
1252114077
1244010265
1193112440
10472
1047212521
10265
3531
3531
4536
4536
5529
5529
4876
4876
6334
6334
7080
7080
7272
7272
7201
7201
6844
6844
4240
4240
9
MS
ud_2u
_test
S
mg_H
H
_2
RP_2
_s
per_s
CN
S
mg_h
h
ug
p_2ug
etr_set
S
mg_h
h
ug
p_2ug
etr_set
7470
7470
B.B.Unique
UniqueProtein
ProteinGroups
GroupsIDs
IDs
4153
4153
4627
4567
4567 4627
4271
4183
4183 4271
4258
4258
3811
3811
2821
2821
2223
2223
1735
1735
8383
5417
5417
UniqueProtein
ProteinGroups
GroupsIDs
IDs
B.B.Unique
2632
2632
2310
2310
2682
2682
2795
2795
2970
2970
3033
3033
2187
2187
1597
1597
4141
73.7
73.7
C.C.Peptide
PeptideFractional
FractionalResolution
Resolution
77.08
77.08
C.C.Peptide
PeptideFractional
FractionalResolution
Resolution
23.8
23.8
6.26.2
3.03.0
19.45
19.45
1.61.6
0.60.6
0.20.2
0.00.0
2.83
2.83
1 fraction
1 fraction 2 fractions
2 fractions 3 fractions
3 fractions 4 fractions
4 fractions 5 fractions
5 fractions 6 fractions
6 fractions 7 fractions
7 fractions 8 fractions
8 fractions
0.51
0.51
0.09
0.09
0.04
0.04
0.01
0.01
0.00
0.00
1 fraction
1 fraction 2 fractions
2 fractions 3 fractions
3 fractions 4 fractions
4 fractions 5 fractions
5 fractions 6 fractions
6 fractions 7 fractions
7 fractions 8 fractions
8 fractions
D.D.Overlaps
Overlapsininunique
uniquepeptide
peptide
identifications
identifications
Conclusions
Conclusions
  High
High
pHpH
reversed-phase
reversed-phase
fractionation
fractionation
of of
complex
complex
sample
sample
digests
digests
leads
leads
to to
higher
higher
numbers
numbers
of of
unique
unique
peptide
peptide
and
and
protein
protein
identifications.
identifications.
  Fractionation
Fractionation
in in
a spin-column
a spin-column
format
format
offers
offers
excellent
excellent
fractional
fractional
resolution
resolution
and
and
reproducibility.
reproducibility.
  Pierce
Pierce
High
pHpH
Reversed-Phase
Fractionation
Fractionation
KitKit
enables
enables
more
more
comprehensive
comprehensive
High
Reversed-Phase
proteomic
proteomic
analysis
analysis
of of
samples
samples
in in
a convenient,
a convenient,
economical,
economical,
and
and
user-friendly
user-friendly
format.
format.
© 2015
© 2015
Thermo
Thermo
Fisher
Fisher
Scientific
Scientific
Inc.Inc.
AllAll
rights
rights
reserved.
reserved.
AllAll
trademarks
trademarks
areare
thethe
property
property
of Thermo
of Thermo
Fisher
Fisher
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its its
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This
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is not
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intended
intended
to encourage
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useuse
of these
of these
products
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in any
in any
manner
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that
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infringe
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of others.
of others.
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