Download Analysis of Subtle Changes in Biological Systems Through use of

Analysis of Subtle Changes in Biological Systems
Through use of High Resolution, High Accuracy
UHPLC Generated Libraries with a Q-Exactive
HF Mass Spectrometer
David A. Sarracino,1 Mauro De Pra, 2 Keely Murphy,1 Jason Neil,1 Mary F. Lopez1
1
Thermo Fisher BRIMS Center, Cambridge, MA, USA; 2Thermo Fisher Scientific, Germering, Germany
O
Overview
i
Purpose: A robust
P
b t method
th d for
f discovering
di
i subtle
btl changes
h
in
i
protein expression in biological systems.
Methods: Using AMAT(Accurate Mass,
Mass Accurate Time)
lib i off peptides
libraries
tid generated
t d from
f
both
b th fractionated
f ti
t d and
d
unfractionated
f ti
t d trypsin
t
i digests
di
t off protein
t i lysates
l
t and
d
biological fluids.
Results: Subtle expression differences in both plasma and
cellll lysates
l
t from
f
th
the model
d l system
t
are revealed
l d using
i the
th
workflow
kfl
described.
d
ib d
FIGURE 1. Vanqu
Introduction
Protein diversity in biological systems is quite large, but short
term (<1hour)
(
) changes
g due to a particular
p
stimulus will cause
only subtle changes in very specific proteins expression
levels Excessive sample handling/processing methods often
levels.
add significant noise to overall experimental results
results. Here we
use a combination of simple sample preparation technique,
technique
extremely reproducible UHPLC system,
system and a robust mass
spectrometer
t
t platform
l tf
to
t afford
ff d researchers
h
the
th ability
bilit to
t use
massive protein/peptide libraries for quantification of several
thousand proteins.
To study the effectiveness of this method,
method a model system
using
i whole
h l live
li bacteria,
b t i both
b th gram negative
ti and
d positive
iti
strains, were spiked into whole blood samples from a single
donor. After 1 hour, plasma and peripheral blood
mononuclear cells were isolated and analyzed
y
for protein
p
expression differences.
The Thermo Scientific™ Vanquish™ UHPLC platform with a
tandem column setup allows for extremely high
reproducibility
p
y across a wide range
g of sample
p loadings,
g ,
independent of sample/matrix type. This allows the use of
upfront sample fractionation to reduce peptide complexity
and hybrid tandem mass spectrums for more confident and
complete library generation,
generation without compromising accurate
retention time information.
information
A
B
FIGURE 2.
2 System
S t
robustness of qua
reproducibility
p
y 20
column. A)) global
g
area and SD comp
isotope area com
Workflo
Input File: SPAD-PBMC-Library-fraction-01.raw
1271
220
955
102
31
318
1984
A
Workflow: SPAD-PBMC-Library-fraction-03
Workflow: SPAD
SPAD-PBMC-Library-fraction-07
PBMC Library fraction 07
M th d
Methods
2
Sample
S
l Preparation
P
ti
Lithium heparin collected blood in plasma tubes, and Cell
Preparation
p
Tubes from an anonymous
y
donor was spiked
p
with buffer,, 50 CFU/mL of E. coli,, or 50 CFU/mL S. Aurus
and allowed to incubate for 1 hour at 37 °C.
C. The samples
were then spun at 2500 RCF for the plasma separation or at
FIGURE 3.
3 Unique
1800 RCF for Peripheral Blood Mononuclear Cell (PBMC)
high pH reverse p
preparations Both the plasma and cells were diluted and
preparations.
l
lysed
d in
i 8M GuHCl
G HCl 250mM
250 M Tris-HCl
T i HCl 5%n-propanol
5%
l 10mM
10 M
Library
ib
Generation
G
i
DTT( H 8
DTT(pH
8.5).
5) Cysteines
C t i
were alkylated
lk l t d with
ith th
the addition
dditi off
In Figure 2 we sho
iodoacetic acid to 45mM. Samples were diluted in digestion
is sta
Analysis of Subtle Changes in Biological Systems
useTris-HCl
of High Resolution,
Accuracyand
UHPLC
Generated
Libraries
withga Q-Exactive quantification
HF Mass Spectrometer
buffer,Through
50mM
5mMHigh
CaCl2,
Pierce
sequencing
q
i
important
t
t
as
lib
library
grade modified trypsin
g
yp
was added. Samples
p
were digested
g
A
complete library generation,
generation without compromising accurate
retention time information.
information
Workflow: SPAD-PBMC-Library-fraction-03
Workflow: SPAD
SPAD-PBMC-Library-fraction-07
PBMC Library fraction 07
M th d
Methods
3266
Sample
S
l Preparation
P
ti
Lithium heparin collected blood in plasma tubes, and Cell
Preparation
p
Tubes from an anonymous
y
donor was spiked
p
with buffer,, 50 CFU/mL of E. coli,, or 50 CFU/mL S. Aurus
and allowed to incubate for 1 hour at 37 °C.
C. The samples
were then spun at 2500 RCF for the plasma separation or at
1800 RCF for Peripheral Blood Mononuclear Cell (PBMC)
preparations Both the plasma and cells were diluted and
preparations.
l
lysed
d in
i 8M GuHCl
G HCl 250mM
250 M Tris-HCl
T i HCl 5%n-propanol
5%
l 10mM
10 M
DTT( H 8
DTT(pH
8.5).
5) Cysteines
C t i
were alkylated
lk l t d with
ith th
the addition
dditi off
iodoacetic acid to 45mM. Samples were diluted in digestion
buffer, 50mM Tris-HCl 5mM CaCl2, and Pierce sequencing
q
g
grade modified trypsin
g
yp
was added. Samples
p
were digested
g
overnight.
C
FIGURE 3.
3 Unique peptid
high pH reverse phase f
Library
ib
Generation
G
i
In Figure 2 we show that
quantification is stable ove
i
important
t t as lib
library ffraction
ti
abundant
b d
peptides.
id
In
I Figu
Fi
fractionation is quite efficie
between fractions.
Liquid Chromatography
Peptide
p
libraries were made byy fractionating
g the peptides
p p
from both the p
plasma and cell lysate
y
samples
p
into 24
fractions on a 4.6mmx250cm PSDVB column 300A pore,
8um particle on a gradient from 99% 50mM ammonium
acetate 0
0.4%
4% ammonium hydroxide/water to 45% 0
0.2%
2%
ammonium hydroxide 10% water/acetonitrile.
water/acetonitrile
FIGURE 4.
4 Even with high
resolution accurate mass
spectrometry,
t
t retention
t ti ti
accuracy is
i important
i
to
assign masses to library
members that may
y not be
intense
te se enough
e oug to ttrigger
gge
events. Three different
samples compared at 3
different mass filter tolera
in a 12 minute window.
window Pe
TEFLSFMNTELAAFTK S1
TEFLSFMNTELAAFTK,
A11 protein.
i
UHPLC was setup with three Thermo Scientific™ Acclaim™
120 C18 3μm 120A 2
2.1mmx250mm
1mmx250mm columns connected by
Thermo Scientific™ Dionex™ Viper™ Fingertight Fitting tubing;
both the solvent pre-heater
pre heater and the column compartment
were sett to
t 55 °C.
°C S
Solvent
l
t A was 0
0.2
2 fformic
i acid
id iin Fi
Fisher
h
O ti
Optima
LC-MS
LC MS grade
d water,
t
S
Solvent
l
t B was 0
0.2
2 fformic
i acid
id
™
™
in Fisher Scientific Optima LC-MS grade acetonitrile. A
gradient of 3-50% over 95minutes at 250 μ
g
μL/min with a total
run length
g of 130 min. A divert valve diverted the solvent for
the first 10 minutes and the last 20 minutes of the gradient.
Pierce Retention Time Calibration standards are added to 1
pmole per injection
injection.
Results
Whole Blood Stimulation
Plasma library generation y
(1% FDR) 6067 peptides w
Mass Spectrometry
p
y
™
A Thermo Scientific Q Exactive™ HF Mass Spectrometer
equipped with a HESI-II
HESI II source with the spray cone installed
was tuned with 35 sheath,
sheath 8 aux,
aux 1 sweep with a vaporizer
temperature of 275 °C
C and a capillary temp of 325 °C
C. The
i t
instrument
t acquisition
i iti iis configured
fi
d with
ith a d
data
t d
dependant
d t
“t 20” method
“top
th d with
ith 350-1500m/z
350 1500 / on the
th full
f ll scan 3e6
3 6 target
t
t
120K resolution, ms/ms are triggered on apex triggers 6-20s
peak width, 2e4 target(100%),
p
g (
) 50ms fill, 15k resolution.
Dynamic
y
exclusion set to 20s,, 25ppm.
pp
PBMC library
yg
generation yyie
53,444
,
p
peptides,
p
, 1072 p
phos
As it would
A
ld be
b a technical
t h i lc
in either one of these samp
from each sample
p can be s
Data
D
t Analysis
A l i
Raw files are searched Proteome Discoverer 1.4 using a
Ch k tto add
Check
dd
simple
p Sequest
q
search template.
p
Parent mass tolerances
All
fil
filtered
d
peptides/proteins
id
/
i
were set to 5ppm,
pp , and fragment
g
mass tolerances were set to
Then add these to the library
15mmu. Plasma and cell lysate data was searched with
static carboxymethyl modified cysteines and differential
oxidized methionines.
methionines Cell lysate data was searched
with Poster Note PN71310 ISC2014-EN 0814S 3
Thermo Scientific
additional phosphorylations on serines,
serines threonines,
threonines and
•
from each sample
p can be
p
peak
width, 2e4 target(100%),
g (
) 50ms fill, 15k resolution.
Dynamic
y
exclusion set to 20s,, 25ppm.
pp
btle Changes
g Data
inA Biological
g
Systems
y
Through
g use o
D t Analysis
l i
files are searched Proteome Discoverer 1.4 using a
UHPLC Generated
G Raw
Lib
with
ithtolerances
a Q Exactive
E
tiCheck
M
Ch k ttoHF
add
dd
simple
p tSequest
qd Libraries
search template.
p i Parent
mass
were set to 5ppm,
pp , and fragment
g
mass tolerances were set to
1 lysate data was searched
1
15mmu. Plasma and cell
with
static carboxymethyl modified cysteines and differential
oxidized methionines.
methionines Cell lysate data was searched with
2
additional phosphorylations on serines,
serines threonines,
threonines and
t
tyrosines
i
with
ith PhosphoRS3.0
Ph
h RS3 0 confirmation
fi
ti node
d sett to
t 0.015
0 015
f
fragment
t ion
i ttolerance.
l
Result
R
lt files(MSF)
fil (MSF) were imported
i
t d into
i t
PinPoint for quantification.
All fil
filtered
d peptides/protein
id /
i
Then add
1 these to the librar
Mauro De Pra2, Keely Murphy , Jason Neil , Mary F.
F Lopez
MS Center
Center, Cambridge
Cambridge, MA
MA, USA , Thermo Fisher Scientific
Scientific, Germer
Comparison
p
of Pro
Selected proteins fro
compared in both the
allowed for direct qu
types as well as acro
S100 protein
t i class
l
w
i f ti models
infection
d l and
d
vering
i subtle
btl changes
h
in
i
ms.
ss,
ss Accurate Time)
both
b th fractionated
f ti
t d and
d
ein
i lysates
l
t and
d
es in both plasma and
re revealed
l d using
i the
th
s is quite large, but short
cular stimulus will cause
proteins expression
rocessing methods often
imental results
results. Here we
preparation technique,
technique
em,
em and a robust mass
archers
h
the
th ability
bilit to
t use
quantification of several
thod,
thod a model system
negative
ti and
d positive
iti
d samples from a single
pheral blood
analyzed
y
for protein
p
UHPLC platform with a
emely high
f sample
p loadings,
g ,
This allows the use of
e peptide complexity
for more confident and
compromising accurate
FIGURE 5. PinPoint lib
to select peptides
p p
by
ym
FIGURE 1. Vanquish UHPLC system and Q Exactive HF MS.
A
Control
Pl
Plasma
E. co
E
Pl
Plasm
C
B
FIGURE 2.
2 System
S t
loading
l di testing,
t ti
showing
h i the
th
robustness of quantification and retention time
reproducibility
p
y 20-200ug
g loading
g of plasma
p
peptides
p p
on
column. A)) global
g
p
protein comparison
p
in PinPoint;; B))
area and SD comparison for replicate injections C)
isotope area comparison
comparison.
FIGURE 6. S100 C
Calprotectin in pla
isotope
p XICs for +
and 2 experimenta
Workflow: SPAD-PBMC-Library-fraction-05
Workflow: SPAD-PBMC-Library-fraction-02
E coli
E.
Workflow:
f
S
SPAD-PBMC-Library-fraction-04
C
f
04
Input File: SPAD-PBMC-Library-fraction-01.raw
1271
2387
220
955
549
2515
102
31
318
S
267
638
90
Control
1984
A
B
3372
Workflow: SPAD-PBMC-Library-fraction-08
SPAD PBMC Library fraction 08
Workflow: SPAD-PBMC-Library-fraction-03
Workflow: SPAD
SPAD-PBMC-Library-fraction-06
PBMC Library fraction 06
Workflow: SPAD
SPAD-PBMC-Library-fraction-07
PBMC Library fraction 07
2673
705
3266
4 Analysis of Subtle Changes in Biological Systems Through use of High Resolution, High Accuracy UHPLC Generated Libraries with a Q-Exactive
A)) HF Mass Spectrometer
557
839
139
ma tubes, and Cell
donor was spiked
p
CFU/mL S. Aurus
°C.
C. The samples
sma separation or at
clear Cell (PBMC)
s were diluted and
n-propanoll 10mM
10 M
with
ith th
the addition
dditi off
e diluted in digestion
Pierce sequencing
q
g
mples
p
were digested
g
ating
g the peptides
p p
mples
p
into 24
olumn 300A pore,
mM ammonium
er to 45% 0
0.2%
2%
nitrile
nitrile.
cientific™ Acclaim™
umns connected by
gertight Fitting tubing;
mn compartment
mic
i acid
id iin Fi
Fisher
h
was 0
0.2
2 fformic
i acid
id
ade acetonitrile. A
0μ
μL/min with a total
erted the solvent for
utes of the gradient.
ards are added to 1
Mass Spectrometer
spray cone installed
FIGURE 6. S100 Calc
Calprotectin in plasm
isotope
p XICs for +2 a
and 2 experimental re
Workflow: SPAD-PBMC-Library-fraction-05
Workflow: SPAD-PBMC-Library-fraction-02
E coli
E.
Workflow:
f
S
SPAD-PBMC-Library-fraction-04
C
f
04
Input File: SPAD-PBMC-Library-fraction-01.raw
1271
2387
220
955
549
2515
102
31
318
S are
S.
267
638
90
Control
1984
A
B
3372
Workflow: SPAD-PBMC-Library-fraction-08
SPAD PBMC Library fraction 08
Workflow: SPAD-PBMC-Library-fraction-03
Workflow: SPAD
SPAD-PBMC-Library-fraction-06
PBMC Library fraction 06
Workflow: SPAD
SPAD-PBMC-Library-fraction-07
PBMC Library fraction 07
2673
705
3266
A))
557
839
139
FIGURE 7.
7 Phosphop
f
from
Serine/Threonin
S i /Th
i
f
from
PBMC lysates
l
A
isotope quantification
3453
C
FIGURE 3.
3 Unique peptides identified in sequential
high pH reverse phase fractions A)1-3
A)1 3 B) 4-6
4 6 C)7
C)7-9.
9
Workflow: SPAD-PBMC-Library-fraction-09
High dynamic range qu
E
Even
without
ith t ttargeted
t d qu
phosphopeptides can be
variability of phosphoryla
replicates
p
is a function o
variability in the system u
simplified sample prepar
workflow affords researc
qualitative and quantitati
systems
systems.
Library
ib
Generation
G
i
In Figure 2 we show that the peptide accuracy and
quantification is stable over a large loading range.
range This is
i
important
t t as lib
library ffractions
ti
will
ill h
have hi
high
h concentrations
t ti
off
abundant
b d
peptides.
id
In
I Figure
Fi
3 we show
h
that
h the
h
fractionation is quite efficient, with minimal peptide overlap
between fractions.
RT: 68.29 - 77.30
FIGURE 4.
4 Even with high
resolution accurate mass
spectrometry,
t
t retention
t ti ti
time
accuracy is
i important
i
to
assign masses to library
members that may
y not be
intense
te se enough
e oug to ttrigger
gge MS2
S
events. Three different
samples compared at 3
different mass filter tolerances
in a 12 minute window.
window Peptide
TEFLSFMNTELAAFTK S100
TEFLSFMNTELAAFTK,
S100,
A11 protein.
i
100
80
70
60
Conclusion
NL: 3.89E6
m/z=
925.4372-925.4742 F:
FTMS + p ESI Full lock
ms [350.00-1500.00]
MS
SPAD-PBMC-Control0101
73.68
71.04
90
71.61
50
40
30
20
10
68.75
68.78
69.38
68.83
68.86
68.68
0
100
69.90 69.93
69.42
70.31
71.73
71.17
70.89
70.41
72.06 72.15
73.56
72.56
72.59
74.25 74.28 74.36 74.57
73.33
72.84
75.13
75.48 75.59 76.05 76.16 76.28
76.69 76.73 77.05
NL: 3.74E6
m/z=
/
925.4372-925.4742 F:
FTMS + p ESI Full lock
ms [350.00-1500.00]
MS
spad-pbmc-control01-02
73.66
73 68
73.68
71.04
90
80
70
Relative Abundance
PLC platform with a
ely high
ample
p loadings,
g ,
s allows the use of
eptide complexity
more confident and
mpromising accurate
FIGURE 2.
2 System
S t
loading
l di testing,
t ti
showing
h i the
th
robustness of quantification and retention time
reproducibility
p
y 20-200ug
g loading
g of plasma
p
peptides
p p
on
column. A)) global
g
p
protein comparison
p
in PinPoint;; B))
area and SD comparison for replicate injections C)
isotope area comparison
comparison.
71.60
20ppm
 Three months of ope
sub 4 second shift in
and samples to be ru
60
50
71.56
40
30
68.77
20
10
0
100
68.73
68.47
71.53
69.94
68.85
69.08 69.35
69.43
69.47
70.00
71.49
72.14 72.21
71.21
70.31 70.39 70.86
72.55
72.61
74.27 74.35 74.41
74.78
73.28 73.45
75.11
75.67
75.44
75.92
76.20
76.48 76.68 76.91 77.11
NL: 3.74E6
m/z=
925.4372-925.4742 F:
FTMS + p ESI Full lock
ms [350.00-1500.00]
MS
spad-pbmc-control02-01
73.67
71.01
90
80
70
71.58 71.61
60
50
40
30
20
68.76
10
0
69.34
69.31
68.82
68.44
68.5
69.0
69.39
69.88
69.43
73.58
69.92
69.97
70.08
69.5
70.0
71.51
71.47
70.88
70.60
70.5
71.0
72.04
71.5
72.13
72.52
72.56
72.49
72.61
72.0
72.5
100
73.26
74.25 74.30 74.37
73.32
73.0
Time (min)
RT: 68.29 - 77.30
73.5
74.0
74.96 75.13 75.32
74.5
75.0
75.67
75.5
76.07
76.15
76.72 76.93 77.28
76.0
76.5
77.0
NL: 3.89E6
m/z=
925.4464-925.4650 F:
FTMS + p ESI Full lock
ms [350.00-1500.00]
MS
SPAD-PBMC-Control0101
73.68
71.04
90
80
70
60
50
40
30
20
10
68.75
68.78
68.83
68.86
68.68
0
100
69.47 69.59 69.65
71.17
70.89
70.12 70.31 70.41
71.31
71.94 72.13 72.47
10ppm
 The only cleaning pe
heated ion transfer c
sweep
p cone after eac
73.56
72.55
72.60
73.84 74.05
73.14 73.39
73.66
73.68
71.04
74.56 74.77 74.90
75.88 76.03 76.22
75.37 75.52
76.70 76.91
77.22
90
80
NL: 3.74E6
m/z=
925.4464-925.4650 F:
FTMS + p ESI Full lock
ms [350.00-1500.00]
MS
spad-pbmc-control01-02
70
Relative Abundance
d,
d a model system
gative
ti and
d positive
iti
mples from a single
ral blood
nalyzed
y
for protein
p
C
B
60
50
40
30
68 77
68.77
20
68.73
10
0
100
68.33
68.85
68.87
69.48 69.63 69.81
71.21
70.20 70.34 70.39
71.72 71.79 71.97
72.50
72.55
72.61
73.84 74.06
72.93 73.24
73.67
71.01
74.44 74.59
75.66 75.92 75.94 76.29
74.94 75.18 75.33
76.90 77.02
NL: 3.74E6
m/z=
925.4464-925.4650 F:
FTMS + p ESI Full lock
ms [350.00-1500.00]
MS
spad-pbmc-control02-01
90
80
70
60
50
40
30
20
0
73.58
68.76
10
68.82
68.49
68.5
69.01
69.0
69.44 69.60
70.01 70.14
69.5
70.0
70.60
70.88
70.5
71.15
71.0
RT: 68.29 - 77.30
71.29
71.5
71.83
72.56 72.58
72.25 72.51
72.73 73.03
72.0
72.5
73.0
Time (min)
73.88 74.05
73.42
73.5
74.0
74.43
74.71
74.5
75.89 76.07 76.24
75.00 75.24 75.35
75.0
75.5
76.0
 R
Raw di
digests
t with
ith no
preparation yielded n
76.75 76.90 76.93
76.5
77.0
NL: 3.70E6
m/z=
925.4511-925.4603 F:
FTMS + p ESI Full lock
ms [350.00-1500.00]
MS
SPAD-PBMC-Control0101
71.04
100
90
80
70
60
50
5ppm
40
30
20
10
68.68 68.82
0
100
70 57
70.57
68 99
68.99
71.17
70.89
72 42
72 30 72.42
72.30
71 31
71.31
71.04
72 74
72.74
73 04 73.16
73.04
73 16
75 37
75.37
76 03
75 90 76.03
75.90
77 19
77 06 77.19
77.06
90
80
NL: 3.67E6
m/z=
925.4511-925.4603 F:
FTMS + p ESI Full lock
ms [350.00-1500.00]
MS
spad-pbmc-control01-02
 Extremely reproducib
measurements of sm
l
low
llevell modified
difi d pe
methods.
70
Relative Abundance
antification of several
60
50
40
30
20
10
0
100
68.33
68.69
68.97
69.63
70.39
71.21 71.33
70.77
71.97
72.42
72.73
72.99
73.24 73.35
75.18 75.36
74.40 74.58
71.01
75.73 75.89
77.30
NL: 3.62E6
m/z=
925.4511-925.4603 F:
FTMS + p ESI Full lock
ms [350.00-1500.00]
MS
spad-pbmc-control02-01
90
80
70
60
50
40
30
20
10
0
68.79 68.82
68.30 68.66
68.95
68.5
69.0
70.55
69.68
69.5
70.0
70.5
70.88
71.15
71.0
72.38 72.43
71.29
71.5
72.0
72.5
73.98
73.03
73.0
Time (min)
73.5
74.0
74.06
76.24
74.79
74.5
75.0
75.5
76.0
77.16
76.5
77.0
Results
Whole Blood Stimulation
Plasma library generation yielded 790 plasma protein groups
(1% FDR) 6067 peptides with +/-10s retention time
accuracy
accuracy.
Thermo Scientific Poster Note
PBMC library
yg
generation yyielded 6200 Protein g
groups(1%
p (
FDR))
References
•
1. Hansen, W. L. J.; Bru
Evaluation of New Pr
PN71310 ISC2014-EN 0814S 5
Tools and DNA Isolat
s at 250 μ
μL/min with a total
alve diverted the solvent for
20 minutes of the gradient.
n standards are added to 1
HF Mass Spectrometer
with the spray cone installed
, 1 sweep with a vaporizer
illary temp of 325 °C
C. The
ed
d with
ith a d
data
t d
dependant
d t
/z
/ on the
th full
f ll scan 3e6
3 6 target
t
t
ered on apex triggers 6-20s
0ms fill, 15k resolution.
5ppm.
pp
™
A11 protein.
i
spad-pbmc-control02-01
70
60
50
40
30
20
10
0
68.79 68.82
68.30 68.66
68.95
68.5
69.0
70.55
69.68
69.5
70.0
70.88
70.5
71.15
71.0
72.38 72.43
71.29
71.5
72.0
73.98
73.03
72.5
73.0
Time (min)
73.5
74.0
74.06
76.24
74.79
74.5
75.0
75.5
76.0
77.16
76.5
77.0
Results
Whole Blood Stimulation
Plasma library generation yielded 790 plasma protein groups
(1% FDR) 6067 peptides with +/-10s retention time accuracy
accuracy.
PBMC library
yg
generation yyielded 6200 Protein g
groups(1%
p (
FDR))
53,444
,
p
peptides,
p
, 1072 p
phosphopeptides(no
p p p
(
specific
p
enrichment))
As it would
A
ld be
b a technical
t h i l challenge
h ll
to
t quantify
tif ALL the
th proteins
t i
in either one of these samples, selected protein and peptides
from each sample
p can be selected from the library.
y
ystems
y
Through
g use of High
g Resolution,
Resolution,
with
ith 1.4
a using
Q aExactive
E
ti HF Mass
M
Spectrometer
S
t
t
e Discoverer
Parent mass tolerances
1
mass tolerances
were set to
data was searched with
steines and differential
data was searched with
erines threonines,
erines,
threonines and
nfirmation
fi
ti node
d sett to
t 0.015
0 015
es(MSF)
(MSF) were imported
i
t d into
i t
Checkk tto add
Ch
dd
All1fil
filtered
d peptides/proteins
id /
i
Then add these to the library
measurement
l
low
llevell mod
d
methods.
References
1. Hansen, W. L
Evaluation of
Tools and DN
Pathogen Det
2009 47,
2009,
47 8,
8 2
http://jcm asm
http://jcm.asm
2. Hao, P.; Ren,
Evaluation of
Interaction Ch
Reversed Pha
off Rat
R t Kidney
Kid
82 254
82,
254—262
262
© 2014 Thermo Fisher Scientific In
Inc. and its subsidiaries. This inform
infringe the intellectual property rig
Neil , Mary F.
F Lopez
ermo Fisher Scientific
Scientific, Germering
Germering, Germany
FIGURE 5. PinPoint library selection tool allows users
to select peptides
p p
by
y mods or proteins
p
by
y name.
Comparison
p
of Proteins in Plasma and PBMC samples
p
Selected proteins from plasma and PBMC libraries were
compared in both the plasma and PBMC samples
samples. This
allowed for direct quantification between the two sample
types as well as across the stimulation conditions.
conditions The
S100 protein
t i class
l
was chosen
h
as it iis wellll represented
t d in
i
i f ti models
infection
d l and
d circulating
i l ti immune
i
cells.
ll
E coli
E.
PBMC
m and Q Exactive HF MS.
S. areus
S
PBMC
Controll
C
PBMC
Control
Pl
Plasma
S areus
E. coli S.
E
Plasma
Pl
Plasma
i
showing
h i the
th
FIGURE 6. S100 Calcium binding protein-A8
etention time
Calprotectin in plasma and PBMC lysates showing top 3
f plasma
p
peptides
p p
on
isotope
p XICs for +2 and +3 charge
g states,, 2 technical
son in PinPoint;; B))
of Subtle Changes in Biologicaland
Systems
Through use of High Resolution,
High Accuracy
UHPLC Generated
6 Analysis C)
2 experimental
replicates
per sample
state.Libraries with a Q-Exactive HF Mass Spectrometer
ate injections
ing
i the
th
on time
ma peptides
p p
on
PinPoint;; B))
ections C)
549
2515
S areus
S.
267
638
90
S. arreuus
S
E coli
E.
Workflow:
f
S
SPAD-PBMC-Library-fraction-04
C
f
04
E. ccoli
E
Conntro
C
ol
FIGURE 6. S100 Calcium binding protein-A8
Calprotectin in plasma and PBMC lysates showing top 3
isotope
p XICs for +2 and +3 charge
g states,, 2 technical
and 2 experimental replicates per sample state.
Control
3372
kflow: SPAD
SPAD-PBMC-Library-fraction-06
PBMC Library fraction 06
B)
A))
FIGURE 7.
7 Phosphopeptide FYAAEIASALGYLHSIK
f
from
Serine/Threonine
S i /Th
i protein
t i kinase
ki
SGK isoform1
i f
1
f
from
PBMC lysates
l
A) area sum off peptides
id B) peptide
id
isotope quantification.
sequential
) 4-6
4 6 C)7
C)7-9.
9
acy and
range
ange. This is
concentrations
t ti
off
at the
h
eptide overlap
NL: 3.89E6
m/z=
925.4372-925.4742 F:
FTMS + p ESI Full lock
ms [350.00-1500.00]
MS
SPAD-PBMC-Control0101
73.68
73.56
74.25 74.28 74.36 74.57
73.33
4
75.13
75.48 75.59 76.05 76.16 76.28
76.69 76.73 77.05
NL: 3.74E6
m/z=
/
925.4372-925.4742 F:
FTMS + p ESI Full lock
ms [350.00-1500.00]
MS
spad-pbmc-control01-02
73.66
73 68
73.68
74.27 74.35 74.41
74.78
73.28 73.45
75.11
75.67
75.44
75.92
76.20
20ppm
76.48 76.68 76.91 77.11
NL: 3.74E6
m/z=
925.4372-925.4742 F:
FTMS + p ESI Full lock
ms [350.00-1500.00]
MS
spad-pbmc-control02-01
73.67
73.58
26
74.25 74.30 74.37
73.32
73.5
74.0
74.96 75.13 75.32
74.5
75.0
75.67
75.5
76.07
76.15
76.72 76.93 77.28
76.0
76.5
77.0
NL: 3.89E6
m/z=
925.4464-925.4650 F:
FTMS + p ESI Full lock
ms [350.00-1500.00]
MS
SPAD-PBMC-Control0101
73.68
10ppm
73.56
73.84 74.05
3.14 73.39
73.66
73.68
73.84 74.06
73.24
73.67
74.56 74.77 74.90
74.44 74.59
75.88 76.03 76.22
75.37 75.52
76.70 76.91
75.66 75.92 75.94 76.29
74.94 75.18 75.33
77.22
NL: 3.74E6
m/z=
925.4464-925.4650 F:
FTMS + p ESI Full lock
ms [350.00-1500.00]
MS
spad-pbmc-control01-02
76.90 77.02
NL: 3.74E6
m/z=
925.4464-925.4650 F:
FTMS + p ESI Full lock
ms [350.00-1500.00]
MS
spad-pbmc-control02-01
High dynamic range quantification
E
Even
without
ith t ttargeted
t d quantification
tifi ti methods,
th d llow llevell
phosphopeptides can be analyzed and quantified.
f
The
variability of phosphorylation amongst experimental
replicates
p
is a function of the modification’s biological
g
variability in the system used, even with the highly
simplified sample preparation techniques used.
used This new
workflow affords researcher a facile method for the rapid
qualitative and quantitative analysis of complex biological
systems.
systems
Conclusion
 Three months of operation on one column setup with
sub 4 second shift in retention time allows for libraries
and samples to be run continuously.
 The only cleaning performed was the swapping of the
heated ion transfer capillary, and washing off the
sweep
p cone after each sample
p set/type.
yp
73.58
3
73.88 74.05
73.42
73.5
74.0
74.43
74.71
74.5
75.89 76.07 76.24
75.00 75.24 75.35
75.0
75.5
76.0
76.75 76.90 76.93
76.5
77.0
NL: 3.70E6
m/z=
925.4511-925.4603 F:
FTMS + p ESI Full lock
ms [350.00-1500.00]
MS
SPAD-PBMC-Control0101
73 04 73.16
73.04
73 16
2.99
75 37
75.37
73.24 73.35
73.98
73.03
73.0
)
73.5
75.18 75.36
74.40 74.58
74.0
74.06
76 03
75 90 76.03
75.90
75.73 75.89
77.30
76.24
75.0
75.5
NL: 3.67E6
m/z=
925.4511-925.4603 F:
FTMS + p ESI Full lock
ms [350.00-1500.00]
MS
spad-pbmc-control01-02
NL: 3.62E6
m/z=
925.4511-925.4603 F:
FTMS + p ESI Full lock
ms [350.00-1500.00]
MS
spad-pbmc-control02-01
74.79
74.5
77 19
77 06 77.19
77.06
5ppm
76.0
77.16
76.5
77.0
a protein groups
n time accuracy
accuracy.
ng
groups(1%
p (
FDR))
specific
p
enrichment))
 R
Raw di
digests
t with
ith no cleanup
l
additional
dditi
l sample
l
preparation yielded no increase in column pressures.
 Extremely reproducible injections allows for
measurements of small biological changes
changes, including
l
low
llevell modified
difi d peptides
tid without
ith t going
i to
t targeted
t
t d
methods.
References
1. Hansen, W. L. J.; Bruggeman,
gg
C. A.; Wolffs, P. F. G.
Evaluation of New Preanalysis
y
Sample
p Treatment
Tools and DNA Isolation Protocols To Improve Bacterial
Thermo Scientific Poster Note
Pathogen Detection in Whole Blood.
Blood Clin.
Clin Microbiol
Microbiol.
2009 47,
2009,
47 8,
8 2629—2631
2629 2631
•
PN71310 ISC2014-EN 0814S 7
70.55
69.68
69.5
70.0
70.5
70.88
71.15
71.0
72.38 72.43
71.29
71.5
72.0
72.5
73.98
73.03
73.0
Time (min)
73.5
74.0
74.06
76.24
74.79
74.5
75.0
75.5
76.0
77.16
76.5
77.0
llow llevell modified
difi d peptides
tid without
ith t going
i to
t targeted
t
t d
methods.
References
plasma protein groups
etention time accuracy
accuracy.
0 Protein g
groups(1%
p (
FDR))
es(no
(
specific
p
enrichment))
o quantify
tif ALL the
th proteins
t i
ed protein and peptides
m the library.
y
1. Hansen, W. L. J.; Bruggeman,
gg
C. A.; Wolffs, P. F. G.
Evaluation of New Preanalysis
y
Sample
p Treatment
Tools and DNA Isolation Protocols To Improve Bacterial
Pathogen Detection in Whole Blood.
Blood Clin.
Clin Microbiol
Microbiol.
2009 47,
2009,
47 8,
8 2629—2631
2629 2631
http://jcm asm org/content/47/8/2629 full
http://jcm.asm.org/content/47/8/2629.full
2. Hao, P.; Ren, Y.; Dutta, B.; Sze, SK. Comparative
Evaluation of Electrostatic Repulsion–Hydrophilic
Repulsion Hydrophilic
Interaction Chromatography (ERLIC) and High
High-pH
pH
Reversed Phase (Hp
(Hp-RP)
RP) Chromatography in Profiling
off Rat
R t Kidney
Kid
Proteome.
P t
J
Journal
l off Proteomics,
P t
i
2013
2013,
82 254
82,
254—262
262
© 2014 Thermo Fisher Scientific Inc. All rights
g
reserved. All trademarks are the p
property
p y 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.
PO71310-EN 0814S
on tool allows users
teins by
y name.
www.thermofisher.com
©2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. 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.
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