Proteored Multicentric Experiment 8 (PME8)
Quantitative Targeted Analysis in Proteomics.
An Assesment Study
(QTAPAS)
ProteoRed WG1-WG2 Meeting
Pamplona, December 10th 2013
Total 27 Participant Laboratories (February- April 2013)
MS
Instrument
Flow rate
LC Column
Gradient
time
Scheduled
VHIO
X
4000QTrap
300nL/min
75 µm x 15 cm
90
no
2
IACS
X
4000QTrap
300 nL/min
75 µm x 15 cm
90
no
3
5500QTrap
15 µl/min
500 µm x 10 cm
20
yes
#
Lab
SRM
1
MPM
ABSciex
X
4
INIBIC
X
5500QTrap
300 nL/min
75 µm x 15 cm
100
no
5
UPF-CRG
X
5500QTrap
300 nL/min
75 µm x 12 cm
35
yes
6
CIMA
X
5500QTrap
300 nL/min
75 µm x 15 cm
90
no
7
CNB
X
5500Qtrap
300 nL/min
75 µm x 15 cm
90
no
8
UCM-PCM
X
5500QTrap
300 nL/min
75 µm x 15 cm
90
no
9
CSPI-Lund
X
TSQ Vantage
300 nL/min
75 µm x 12 cm
60
yes
X
TSQ Vantage
300 nL/min
75 µm x 15 cm
30
yes
X
TSQ Vantage
300 nL/min
75 µm x 15 cm
60
yes
10
11
Groningen
12
X
TSQ Vantage
300 nL/min
yes
UV
X
5600TripleTOF
300 nL/min
75 µm x 15 cm
75 µm x 12.3
cm
90
13
90
no
14
Navarra Bio.
X
5500QTrap
300 nL/min
75 µm x 15 cm
90
no
15
Sigma
X
5500QTrap
4 µL/min
500 µm x 15 cm
45
no
16
UA
X
Agilent 6490
300 nL/min
75 µm x 15 cm
90
yes
17
Turku
X
TSQVantage
300 nL/min
75 µm x 15 cm
90
yes
X
4000 QTrap
300 nL/min
75 µm x 15 cm
90
no
X
Agilent 6490
300 nL/min
75 µm x 15 cm
90
no
X
5500QTrap
250 µL/min
75 µm x 15 cm
90
no
18
19
UVIC
20
UiB
21
EHU
X
Q-Exactive
300 nL/min
50 µm x 15 cm
90
22
IRBB
X
Synapt G1
250 nL/min
75 µm x 25 cm
90
23
CIB
X
Orbitrap Velos
250 nL/min
75 µm x 15 cm
90
24
X
Orbitrap Velos
300 nL/min
75 µm x 10 cm
90
25
CBMSO
CICSalamanca
X
Orbitrap Velos
400 nL/min
75 µm x 10 cm
90
26
LPCSIC-UAB
X
Orbitrap Velos
0.8 µL/min
100 µm x 15 cm
90
27
CCiT-UB
X
Orbitrap Velos
400 nL/min
75 µm x 10 cm
90
28
PCB
X
Orbitrap Velos
250 µL/min
75 µm x 25 cm
90
29
CMU-Geneva
X
Orbitrap Velos
250 µL/min
75 µm x 25 cm
90
30
CICbioGUNE
X
Orbitrap XL ETD
300 nL/min
75 µm x 20 cm
90
20 SRM data sets
10 PRM data sets:
3 QTOF
7 OT
TQ1
TQ2
TQ3
TQ4
TQ5
TQ6
TQ7
TQ8
TQ9
TQ10
TQ11
TQ12
TQ13
TQ14
TQ15
TQ16
TQ17
TQ18
TQ19
TQ20
QTOF21
QTOF22
QTOF23
OT24
OT25
OT26
OT27
OT28
OT29
OT30
VHIO
IACS
UVIC1 (Vancouver)
AbSciex
INIBIC
UPF-CRG
CIMA
CNB
UCM-PCM
SIGMA
UiB(Bergen)
NavarraBiomed
UA
UVIC2 (Vancouver)
Turku
CSPI (Lund)
Groningen 30
Groningen 60
Groningen 90
Waters
UV
EHU
IRB
CIB
CBMSO
CIC-Salamanca
CCiT
PCB
LPCSIC-UAB
CiCBiogune
MRM
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
PRM
x
x
x
x
x
x
x
x
x
x
Mass Spectrometer
4000QTrap
4000QTrap
4000QTrap
5500QTrap
5500QTrap
5500QTrap
5500QTrap
5500Qtrap
5500QTrap
5500QTrap
5500QTrap
5500QTrap
Agilent 6490 QQQ
Agilent 6490 QQQ
TSQ Vantage
TSQ Vantage
TSQ Vantage
TSQ Vantage
TSQ Vantage
Xevo TQ-S
5600TripleTOF
Q-Exactive
Waters Synapt G1
LTQ Orbitrap Velos
LTQ Orbitrap Velos
LTQ Orbitrap Velos
LTQ Orbitrap Velos
LTQ Orbitrap Velos
LTQ Orbitrap Velos
Orbitrap XL ETD
TQ
QTOF
OT
SIGMA-ALDRICH MSQC1
- 6 Digested proteins :
25-fold concentration range – 3 concentration tiers
Quantified UV before digestion
- 2-3 labeled peptides/ protein
L/H ratios 0.2-50
Quantified AAA
SAMPLE SET
Yeast Digest + Spiked MSQC1, 5 different concentrations
Tier Protein
CAH1
1 CAH2
NADPH
2 CRP
PPIA
3 CATA
fmol / mg yeast digest
sample A sample B sample C sample D sample E
20
40
100
200
1000
20
40
100
200
1000
4
8
20
40
200
4
8
20
40
200
0.8
1.6
4
8
40
0.8
1.6
4
8
40
fmol / microgram Yeast Lysate Digest
LABELED PEPTIDES
Tier
1
2
3
ratio L/H
CAH1_GGPFSDSY[R]
1
Labeled
AQUA Peptides
fmol/m g Yeast digest
CAH1_VLDALQAI[K]
2
CAH2_AVQQPDGLAVLGIFL[K]
10
A
B
C
CAH2_SADFTNFDP[R]
50
NADPH_EGHLSPDIVAEQ[K]
1
GGPFSDSY[R]
20
40
CAH1
VLDALQAI[K]
10
20
NADPH_ALIVLAHSE[R]
2
AVQQPDGLAVLGIFL[K]
2
4
CRP_ESDTSYVSL[K]
10
CAH2
SADFTNFDP[R]
0.4
0.8
CRP_GYSIFSYAT[K]
50
EGHLSPDIVAEQ[K] 0.5
4
8
PPIA_FEDENFIL[K]
NADPH
ALIVLAHSE[R]
2
4
PPIA_VSFELFAD[K]
1
ESDTSYVSL[K]
0.4
0.8
PPIA_TAENF[R]
2
CRP
GYSIFSYAT[K]
0.08
0.16
CATA_GAGAFGYFEVTHDIT[K]
0.2
FEDENFIL[K]
1.6
3.2
CATA_FSTVAGESGSADTV[R]
10
PPIA
VSFELFAD[K]
0.8
1.6
CATA_NLSVEDAA[R]
50
TAENF[R]
0.4
0.8
GAGAFGYFEVTHDIT[K]
4
8
CATA
FSTVAGESGSADTV[R]
0.08
0.16
NLSVEDAA[R]
0.016
0.032
Peptide
D
100
50
10
2
20
10
2
0.4
8
4
2
20
0.4
0.08
E
200
1000
100
500
20
100
4
20
40
200
20
100
4
20
0.8
4
16
80
8
40
4
20
40
200
0.8
4
0.16
0.8
QTAPAS - PME8 SRM ANALYSIS
RECOMMENDED GUIDELINES
1- LC conditions:
-Nano LC- system:
Column: 75 mm x 15 cm C18
Sample load: 1 mg yeast digest/injection
Flow rate: 300-500 nL /min
Gradient: 0-35% Acetonitrile in 90 min
3- Analysis design:
- Samples A through E Replica 1 (from most diluted
to most concentrated)
- Blank runs (enough to secure clean baseline)
- Samples A through E Replica 2
- Blank runs
- Samples A through E Replica 3
http://www.proteored.org/PME8_main.asp
LINEARITY OF RESPONSE
Average of 30 datasets – Error bars: Std Dev.
Tier 1
CAH1
CAH1
10000
1200
y = 1.0153x
R² = 1
800
1000
y = 0.9987x
R² = 1
600
400
log measured fmol
measured fmol
CAH1_GGPFSDSYR
CAH1_VLDALQAIK
CAH1_GGPFSDSYR
CAH1_VLDALQAIK
1000
100
10
200
1
0
0
200
400
600
theoretical fmol
800
1000
1
10
100
log theoretical fmol
1000
10000
Tier 2
NADPH
NADPH
1000
200
NADPH_EGHLSPDIVAEQK
NADPH_ALIVLAHSER
y = 0.8673x
R² = 0.9998
120
80
y = 0.4689x
R² = 0.9999
40
log measured fmol
measured fmol
160
NADPH_EGHLSPDIVAEQK
NADPH_ALIVLAHSER
100
10
1
0
0
40
80
120
theoretical fmol
160
200
1
10
100
log theoretical fmol
1000
Tier 3
PPIA
PPIA
30
100
25
y = 0.5552x
R² = 0.9999
20
15
log measured fmol
measured fmol
PPIA_FEDENFILK
PPIA_VSFELFADK
PPIA_FEDENFILK
PPIA_VSFELFADK
y = 0.5579x
R² = 0.9993
10
10
1
0.1
1
10
5
0
0
0
5
10
15
20
25
30
theoretical fmol
35
40
45
log theoretical fmol
100
ABSOLUTE QUANTIFICATION
INTER LABORATORY VARIABILITY
%CV 30 datasets
Average of 30 datasets , three replicas averaged
Sample
A
Peptide
theor. fmol
ALL MEASUREMENTS
B
C
D
E
A
B
Average fmol measured
C
D
E
CV(%)
20
40
100
200
1000
CAH1_GGPFSDSYR
20.41
40.65
102.30
202.87
1015.23
4.80
6.29
4.81
4.90
4.82
CAH1_VLDALQAIK
20.58
41.02
96.75
201.47
998.66
13.19
6.02
12.89
6.93
10.74
CAH2_AVQQPDGLAVLGIFLK
16.93
33.76
87.94
154.83
852.65
45.22
28.31
27.13
19.93
10.87
CAH2_SADFTNFDPR
19.58
41.44
107.41
221.13
1125.46
19.80
19.19
18.15
10.02
12.65
4.00
8.00
20.00
40.00
200.00
NADPH_EGHLSPDIVAEQK
theor. fmol
3.66
7.15
19.41
35.38
173.10
27.02
20.71
20.90
13.50
11.60
NADPH_ALIVLAHSER
1.85
3.76
9.25
19.35
93.68
22.78
15.78
15.45
11.50
7.86
CRP_ESDTSYVSLK
3.92
7.87
18.43
36.81
185.79
22.05
28.90
9.89
6.88
7.42
CRP_GYSIFSYATK
2.16
4.47
13.32
28.76
149.52
47.30
27.58
13.06
14.66
9.00
0.80
1.60
4.00
8.00
40.00
PPIA_FEDENFILK
0.50
0.93
2.23
4.33
22.23
18.92
18.67
22.08
12.87
15.91
PPIA_VSFELFADK
0.72
1.02
2.59
4.48
22.27
48.45
37.92
28.81
14.34
14.65
CATA_GAGAFGYFEVTHDITK
1.15
0.53
1.47
2.56
8.29
75.82
48.13
67.03
45.56
24.25
CATA_FSTVAGESGSADTVR
0.55
0.92
2.49
4.69
22.13
113.34
41.87
33.56
18.53
11.48
CATA_NLSVEDAAR
0.27
0.63
2.33
4.96
27.22
79.90
61.85
41.77
30.83
12.96
theor. fmol
ABSOLUTE QUANTIFICATION
INTER LABORATORY VARIABILITY
Sample
A
Peptide
theor. fmol
A
B
C
D
E
CV(%)
20
40
100
200
1000
20.50
40.96
102.29
204.21
1024.68
5.42
5.31
5.26
5.33
5.38
CAH1_VLDALQAIK
21.01
41.29
99.34
205.31
1004.96
13.47
5.82
12.17
4.68
9.64
CAH2_AVQQPDGLAVLGIFLK
15.71
33.27
89.06
168.98
844.86
40.58
24.68
25.32
9.58
9.14
CAH2_SADFTNFDPR
20.69
44.12
114.38
228.20
1154.97
11.26
13.75
9.99
4.11
4.57
4.00
8.00
20.00
40.00
200.00
NADPH_EGHLSPDIVAEQK
3.80
7.38
19.10
35.85
173.37
24.82
19.31
15.22
12.91
12.23
NADPH_ALIVLAHSER
2.07
3.89
9.55
19.59
95.10
13.57
16.00
15.81
12.06
6.00
CRP_ESDTSYVSLK
3.83
7.40
18.79
37.06
187.42
17.24
10.46
6.62
5.15
6.82
CRP_GYSIFSYATK
2.17
4.38
13.17
29.62
149.62
52.60
30.63
13.75
13.10
9.50
0.80
1.60
4.00
8.00
40.00
PPIA_FEDENFILK
0.51
0.90
2.15
4.29
21.32
18.58
16.83
13.64
13.98
9.71
PPIA_VSFELFADK
0.72
1.06
2.38
4.47
22.30
41.29
31.94
19.19
15.42
15.93
CATA_GAGAFGYFEVTHDITK
1.12
0.47
1.24
2.25
8.04
80.74
46.49
79.39
46.25
25.30
CATA_FSTVAGESGSADTVR
0.62
0.90
2.27
4.79
22.02
113.39
18.95
23.42
17.60
12.70
CATA_NLSVEDAAR
0.30
0.69
2.24
4.95
27.12
76.51
59.91
44.70
33.85
8.64
A
B
A
B
C
D
theor. fmol
Sample
Peptide
theor. fmol
ORBITRAP
C
D
E
Average fmol measured
E
CV(%)
20
40
100
200
1000
CAH1_GGPFSDSYR
20.01
39.82
101.79
198.71
1001.52
1.84
10.81
4.09
3.48
1.37
CAH1_VLDALQAIK
18.22
39.73
86.40
181.99
986.23
11.41
9.08
11.49
10.75
11.83
CAH2_AVQQPDGLAVLGIFLK
19.66
29.31
73.73
124.25
934.06
65.94
29.58
17.19
29.67
17.50
CAH2_SADFTNFDPR
15.13
29.72
90.46
186.49
1002.49
37.49
18.12
29.96
24.88
27.64
4.00
8.00
20.00
40.00
200.00
NADPH_EGHLSPDIVAEQK
3.06
5.79
22.60
27.55
161.07
10.04
21.75
53.26
NADPH_ALIVLAHSER
1.33
3.29
8.59
18.44
88.68
17.52
7.65
13.20
10.16
17.06
CRP_ESDTSYVSLK
3.69
7.49
15.61
32.95
167.97
9.04
49.12
8.07
17.33
9.53
CRP_GYSIFSYATK
1.97
4.54
14.65
25.03
theor. fmol
N=7
E
Average fmol measured
CAH1_GGPFSDSYR
theor. fmol
N=20
TRIPLE-QUADRUPOLE
C
D
B
theor. fmol
15.11
0.80
1.60
4.00
8.00
40.00
PPIA_FEDENFILK
0.41
0.96
2.05
4.48
24.72
6.74
18.29
10.26
5.88
24.20
PPIA_VSFELFADK
0.83
0.97
2.96
4.66
22.04
70.22
57.51
41.07
14.31
14.98
CATA_GAGAFGYFEVTHDITK
0.79
0.45
1.56
2.40
9.19
56.87
14.18
73.57
29.09
21.71
CATA_FSTVAGESGSADTVR
0.37
0.80
2.98
3.94
22.38
12.72
9.71
41.03
25.04
8.84
CATA_NLSVEDAAR
0.14
0.40
2.91
5.30
24.86
109.32
71.70
32.27
10.70
21.96
ABSOLUTE QUANTIFICATION
INTER LABORATORY VARIABILITY
Sample
A
Peptide
theor. fmol
N=20
E
A
B
Average fmol measured
C
D
E
CV(%)
20
40
100
200
1000
CAH1_GGPFSDSYR
20.50
40.96
102.29
204.21
1024.68
5.42
5.31
5.26
5.33
5.38
CAH1_VLDALQAIK
21.01
41.29
99.34
205.31
1004.96
13.47
5.82
12.17
4.68
9.64
CAH2_AVQQPDGLAVLGIFLK
15.71
33.27
89.06
168.98
844.86
40.58
24.68
25.32
9.58
9.14
CAH2_SADFTNFDPR
20.69
44.12
114.38
228.20
1154.97
11.26
13.75
9.99
4.11
4.57
4.00
8.00
20.00
40.00
200.00
NADPH_EGHLSPDIVAEQK
3.80
7.38
19.10
35.85
173.37
24.82
19.31
15.22
12.91
12.23
NADPH_ALIVLAHSER
2.07
3.89
9.55
19.59
95.10
13.57
16.00
15.81
12.06
6.00
CRP_ESDTSYVSLK
3.83
7.40
18.79
37.06
187.42
17.24
10.46
6.62
5.15
6.82
CRP_GYSIFSYATK
2.17
4.38
13.17
29.62
149.62
52.60
30.63
13.75
13.10
9.50
0.80
1.60
4.00
8.00
40.00
PPIA_FEDENFILK
0.51
0.90
2.15
4.29
21.32
18.58
16.83
13.64
13.98
9.71
PPIA_VSFELFADK
0.72
1.06
2.38
4.47
22.30
41.29
31.94
19.19
15.42
15.93
CATA_GAGAFGYFEVTHDITK
1.12
0.47
1.24
2.25
8.04
80.74
46.49
79.39
46.25
25.30
CATA_FSTVAGESGSADTVR
0.62
0.90
2.27
4.79
22.02
113.39
18.95
23.42
17.60
12.70
CATA_NLSVEDAAR
0.30
0.69
2.24
4.95
27.12
76.51
59.91
44.70
33.85
8.64
Sample
A
B
C
D
E
theor. fmol
theor. fmol
Peptide
theor. fmol
Q-TOF
C
D
E
A
B
Average fmol measured
CV(%)
20
40
100
200
1000
CAH1_GGPFSDSYR
20.44
40.20
102.97
200.38
981.40
3.27
3.00
3.97
3.47
2.71
CAH1_VLDALQAIK
21.19
41.07
95.07
204.44
977.43
3.65
1.80
14.84
4.31
19.57
CAH2_AVQQPDGLAVLGIFLK
19.40
43.28
107.15
139.73
792.83
7.23
30.28
32.62
27.68
4.19
CAH2_SADFTNFDPR
20.71
43.44
90.52
212.91
1182.04
21.38
9.12
31.40
0.71
11.31
58.66
23.84
16.67
13.41
6.86
theor. fmol
N=3
TRIPLE-QUADRUPOLE
C
D
B
4.00
8.00
20.00
40.00
200.00
NQO1_EGHLSPDIVAEQK
3.63
7.54
18.53
35.77
179.64
NQO1_ALIVLAHSER
1.68
3.56
8.28
18.56
89.12
CRP_ESDTSYVSLK
4.86
11.05
19.15
37.98
188.45
48.40
37.45
CRP_GYSIFSYATK
2.13
4.96
14.12
24.56
148.97
16.25
18.92
0.80
1.60
4.00
8.00
40.00
PPIA_FEDENFILK
0.51
1.14
3.16
4.65
24.84
PPIA_VSFELFADK
0.42
0.94
3.32
4.26
22.47
CATA_GAGAFGYFEVTHDITK
2.70
1.10
2.25
5.01
CATA_FSTVAGESGSADTVR
0.28
1.16
3.54
5.02
22.84
CATA_NLSVEDAAR
0.28
0.75
1.81
4.39
31.48
theor. fmol
3.04
24.77
15.39
3.18
5.60
7.70
21.92
7.51
39.59
25.76
7.97
97.18
93.92
49.89
14.23
0.20
20.08
fmol measured
TQ
Sample
OT
QTOF
D
E
D
E
D
E
200
1000
200
1000
200
1000
CAH1_GGPFSDSYR
204.21
1024.68
198.71
1001.52
200.38
981.40
CAH1_VLDALQAIK
205.31
1004.96
181.99
986.23
204.44
977.43
CAH2_AVQQPDGLAVLGIFLK
168.98
844.86
124.25
934.06
139.73
792.83
CAH2_SADFTNFDPR
228.20
1154.97
186.49
1002.49
212.91
1182.04
theor. fmol
40.00
200.00
40.00
200.00
40.00
200.00
NADPH_EGHLSPDIVAEQK
35.85
173.37
27.55
161.07
35.77
179.64
NADPH_ALIVLAHSER
19.59
95.10
18.44
88.68
18.56
89.12
CRP_ESDTSYVSLK
37.06
187.42
32.95
167.97
37.98
188.45
CRP_GYSIFSYATK
29.62
149.62
25.03
24.56
148.97
8.00
40.00
8.00
40.00
8.00
40.00
PPIA_FEDENFILK
4.29
21.32
4.48
24.72
4.65
24.84
PPIA_VSFELFADK
4.47
22.30
4.66
22.04
4.26
22.47
CATA_GAGAFGYFEVTHDITK
2.25
8.04
2.40
9.19
5.01
CATA_FSTVAGESGSADTVR
4.79
22.02
3.94
22.38
5.02
22.84
CATA_NLSVEDAAR
4.95
27.12
5.30
24.86
4.39
31.48
Peptide
theor. fmol
theor. fmol
%CV
TQ
Sample
OT
QTOF
D
E
D
E
D
E
200
1000
200
1000
200
1000
CAH1_GGPFSDSYR
5.33
5.38
3.48
1.37
3.47
2.71
CAH1_VLDALQAIK
4.68
9.64
10.75
11.83
4.31
19.57
CAH2_AVQQPDGLAVLGIFLK
9.58
9.14
29.67
17.50
27.68
4.19
CAH2_SADFTNFDPR
4.11
4.57
24.88
27.64
0.71
11.31
40.00
200.00
40.00
200.00
40.00
200.00
NADPH_EGHLSPDIVAEQK
12.91
12.23
15.11
13.41
6.86
NADPH_ALIVLAHSER
12.06
6.00
10.16
17.06
CRP_ESDTSYVSLK
5.15
6.82
17.33
9.53
CRP_GYSIFSYATK
13.10
9.50
8.00
40.00
8.00
40.00
PPIA_FEDENFILK
13.98
9.71
5.88
24.20
25.76
PPIA_VSFELFADK
15.42
15.93
14.31
14.98
7.97
CATA_GAGAFGYFEVTHDITK
46.25
25.30
29.09
21.71
CATA_FSTVAGESGSADTVR
17.60
12.70
25.04
8.84
CATA_NLSVEDAAR
33.85
8.64
10.70
21.96
Peptide
theor. fmol
theor. fmol
theor. fmol
3.18
5.60
7.70
21.92
7.51
8.00
40.00
14.23
0.20
20.08
ABSOLUTE QUANTIFICATION
Comparison by type of measurement
Sample E (1000 fmol)
200
950
25
24
fmol measured
1100
1000
CATA_FSTVAGESGSADTVR
NADPH_EGHLSPDIVAEQK
220
fmol measured
fmol measured
CAH1_GGPFSDSYR
1150
1050
Sample E (40 fmol)
Sample E (200 fmol)
180
160
140
900
OT
QTOF
21
19
TQ
Sample A (20 fmol)
22
20
120
TQ
23
OT
TQ
QTOF
CAH1_GGPFSDSYR
CATA_FSTVAGESGSADTVR
NADPH_EGHLSPDIVAEQK
7
22
6
QTOF
Sample A (0.8 fmol)
Sample A (4 fmol)
23
OT
0.7
0.6
20
fmol measured
fmol measured
fmol measured
0.5
21
5
4
0.4
0.3
0.2
19
3
0.1
18
0
2
TQ
OT
QTOF
TQ
OT
QTOF
TQ
OT
QTOF
ABSOLUTE QUANTIFICATION
INTRA- vs INTER LABORATORY VARIABILITY
Average, %CV 3 replicas
-
Average, %CV 30 datasets
30
Sample E
25
%CV
20
15
10
Inter-Lab %CV
Intra-Lab %CV
5
0
INTRA- vs INTER LABORATORY VARIABILITY
Average, %CV 3 replicas
120
-
Average, %CV 30 datasets
Sample A
100
%CV
80
60
40
Inter-Lab %CV
Intra-Lab %CV
20
0
REPRODUCIBILITY OF TRANSITION PATTERN
Transition signal distribution
Average 3 replicas
1.2
CAH1_VLDALQAIK
1
Normalized dot product to average distribution
1.02
CAH1_VLDALQAI[K]
1
0.8
TQ B
0.98
0.6
QTOF B
OT B
0.96
TQ E
0.4
0.94
QTOF E
0.2
OT E
0.92
0
0.9
1.2
1
0.8
CATA_FSTVAGESGSADTVR
CATA_FSTVAGESGSADTVR
1
0.95
TQ B
QTOF B
0.6
OT B
0.9
0.4
0.2
0
TQ E
QTOF E
0.85
0.8
OT E
REPRODUCIBILITY OF TRANSITION PATTERN
1.2
CAH2_AVQQPDGLAVLGIFLK
1
1
0.9
0.8
0.8
0.6
0.4
0.2
0
CAH2_AVQQPDGLAVLGIFL[K]
0.7
TQ E
0.6
QTOF E
0.5
OT E
0.4
0.3
0.2
INTRA- vs INTER LABORATORY VARIABILITY
120
Sample A
100
%CV
80
60
40
Inter-Lab %CV
Intra-Lab %CV
20
0
Analysis of pure MSQC1 (without yeast
background)
For comparison: Possible effects of complex
background
3 Vials x 10 mg
200 ng injection – 80 fmol min
Proteored Multicentric Experiment 8: Quantitative Targeted
Analysis in Proteomics. An Assessment Study. (PME8-QTAPAS)
Francesc Canals1 , Joan-Josep Bech-Serra1, Núria Colomé-Calls1, Salvador Martínez-Bartolomé2, Jim J. Walters3, Kevin B. Ray3,
Juan-Pablo Albar2, ProteoRed-ISCIII Consortium4
1 Proteomics Laboratory,
Vall d’Hebron Institute of Oncology (VHIO) Vall d’Hebron University Hospital, 08035 Barcelona, Spain
Centro Nacional de Biotecnologia-CSIC, Madrid, Spain
Sigma-Aldrich, St. Louis, MO
4 ProteoRed-ISCIII Consortium. Spanish Networked Proteomics Platform
fcanals@vhio.net ; jpalbar@proteored.org
2
3
SAMPLE SET
STUDY DESIGN AND OBJECTIVE
Analysis conditions
- 3 replicate nLC-MS runs, 90 min gradient , batchwise (3 x [A-E ])
- SRM: Monitoring (14 +14) peptides x 3 transitions = 72 transitions
- MPM: Monitoring (14+14) peptides, 30 precursor ions
Participant Laboratories
fmol / mg yeast digest
sample A sample B sample C sample D sample E
20
40
100
200
1000
20
40
100
200
1000
4
8
20
40
200
4
8
20
40
200
0.8
1.6
4
8
40
0.8
1.6
4
8
40
Tier Protein
CAH1
1 CAH2
NADPH
2 CRP
PPIA
3 CATA
Tier
Peptide
CAH1_GGPFSDSY[R]
CAH1_VLDALQAI[K]
1
CAH2_AVQQPDGLAVLGIFL[K]
CAH2_SADFTNFDP[R]
NADPH_EGHLSPDIVAEQ[K]
NADPH_ALIVLAHSE[R]
2
CRP_ESDTSYVSL[K]
CRP_GYSIFSYAT[K]
PPIA_FEDENFIL[K]
PPIA_VSFELFAD[K]
• Set of 5 different samples A-E, prepared by spiking
different amounts of the Sigma-Aldrich MSQC1 standard
into a yeast lysate digest.
• The samples contain tryptic digests of 6 human proteins,
distributed in three concentration tiers, as shown in the
table. Amounts indicated as fmol spiked protein/
microgram of yeast lysate
• Additionally, the samples contain isotopically labeled
peptides for each of the human proteins, in different ratios
to the corresponding unlabeled peptides, ranging form
1:0.2 to 1:50, as indicated in the table.
PPIA_TAENF[R]
3
CATA_GAGAFGYFEVTHDIT[K]
CATA_FSTVAGESGSADTV[R]
CATA_NLSVEDAA[R]
ratio L/H
1
2
10
50
1
2
10
50
0.5
1
2
0.2
10
50
• 20 SRM Triple Quadrupole Data Sets
• 3 Q-TOF + 7 ORBITRAP MPM Data Sets
INTRA- LABORATORY VARIABILITY
RESULTS. ABSOLUTE QUANTIFICATION. INTER- LABORATORY/MEASUREMENT TYPE VARIABILITY
A
B
TRIPLE-QUADRUPOLE
C
D
E
A
B
5.42
5.31
C
Average fmol measured
D
E
5.33
5.38
Sample
CV(%)
20
40
100
200
1000
20.50
40.96
102.29
204.21
1024.68
A
B
Peptide
30
ALL MEASUREMENTS
C
D
E
A
B
4.80
6.29
C
Average fmol measured
theor. fmol
5.26
CAH1_GGPFSDSYR
Sample E
D
E
4.90
4.82
CV(%)
20
40
100
200
1000
20.41
40.65
102.30
202.87
1015.23
25
4.81
CAH1_VLDALQAIK
21.01
41.29
99.34
205.31
1004.96
13.47
5.82
12.17
4.68
9.64
CAH1_VLDALQAIK
20.58
41.02
96.75
201.47
998.66
13.19
6.02
12.89
6.93
10.74
CAH2_AVQQPDGLAVLGIFLK
15.71
33.27
89.06
168.98
844.86
40.58
24.68
25.32
9.58
9.14
CAH2_AVQQPDGLAVLGIFLK
16.93
33.76
87.94
154.83
852.65
45.22
28.31
27.13
19.93
10.87
CAH2_SADFTNFDPR
20.69
44.12
114.38
228.20
1154.97
11.26
13.75
9.99
4.11
4.57
CAH2_SADFTNFDPR
19.58
41.44
107.41
221.13
1125.46
19.80
19.19
18.15
10.02
12.65
24.82
12.23
NADPH_EGHLSPDIVAEQK
27.02
11.60
theor. fmol
NADPH_EGHLSPDIVAEQK
4.00
8.00
20.00
40.00
200.00
3.80
7.38
19.10
35.85
173.37
15.22
12.91
9.55
19.59
95.10
13.57
16.00
15.81
12.06
18.79
37.06
187.42
17.24
10.46
6.62
5.15
4.38
13.17
29.62
149.62
52.60
30.63
13.75
13.10
1.60
4.00
8.00
40.00
2.07
3.83
CRP_GYSIFSYATK
theor. fmol
theor. fmol
19.31
3.89
7.40
2.17
0.80
NADPH_ALIVLAHSER
CRP_ESDTSYVSLK
4.00
8.00
20.00
40.00
200.00
3.66
7.15
19.41
35.38
173.10
20.71
20.90
13.50
3.76
9.25
19.35
93.68
22.78
15.78
15.45
11.50
7.87
18.43
36.81
185.79
22.05
28.90
9.89
6.88
2.16
4.47
13.32
28.76
149.52
47.30
27.58
13.06
14.66
0.80
1.60
4.00
8.00
40.00
6.00
NADPH_ALIVLAHSER
1.85
6.82
CRP_ESDTSYVSLK
3.92
9.50
CRP_GYSIFSYATK
theor. fmol
7.86
0.90
2.15
4.29
21.32
18.58
16.83
13.64
13.98
9.71
PPIA_FEDENFILK
0.50
0.93
2.23
4.33
22.23
18.92
18.67
22.08
12.87
15.91
1.06
2.38
4.47
22.30
41.29
31.94
19.19
15.42
15.93
PPIA_VSFELFADK
0.72
1.02
2.59
4.48
22.27
48.45
37.92
28.81
14.34
14.65
CATA_GAGAFGYFEVTHDITK
1.12
0.47
1.24
2.25
8.04
80.74
46.49
79.39
46.25
25.30
CATA_GAGAFGYFEVTHDITK
1.15
0.53
1.47
2.56
8.29
75.82
48.13
67.03
45.56
24.25
CATA_FSTVAGESGSADTVR
0.62
0.90
2.27
4.79
22.02
113.39
18.95
23.42
17.60
12.70
CATA_FSTVAGESGSADTVR
0.55
0.92
2.49
4.69
22.13
113.34
41.87
33.56
18.53
11.48
CATA_NLSVEDAAR
0.30
0.69
2.24
4.95
27.12
76.51
59.91
44.70
33.85
8.64
CATA_NLSVEDAAR
0.27
0.63
2.33
4.96
27.22
79.90
61.85
41.77
30.83
12.96
B
ORBITRAP
C
1000
1001.52
B
10.81
C
4.09
D
Sample E (1000 fmol)
11.41
9.08
11.49
10.75
11.83
65.94
29.58
17.19
29.67
17.50
90.46
186.49
1002.49
37.49
18.12
29.96
24.88
27.64
10.04
21.75
53.26
4.00
8.00
20.00
40.00
200.00
3.06
5.79
22.60
27.55
161.07
15.11
NADPH_ALIVLAHSER
1.33
3.29
8.59
18.44
88.68
17.52
7.65
13.20
10.16
17.06
CRP_ESDTSYVSLK
3.69
7.49
15.61
32.95
167.97
9.04
49.12
8.07
17.33
9.53
1.97
4.54
14.65
25.03
0.80
1.60
4.00
8.00
PPIA_FEDENFILK
0.41
0.96
PPIA_VSFELFADK
0.83
0.97
40.00
2.05
4.48
24.72
2.96
4.66
22.04
6.74
18.29
10.26
5.88
24.20
70.22
57.51
41.07
14.31
14.98
56.87
14.18
73.57
29.09
21.71
9.71
41.03
25.04
8.84
71.70
32.27
10.70
21.96
CATA_GAGAFGYFEVTHDITK
0.79
0.45
1.56
2.40
9.19
CATA_FSTVAGESGSADTVR
0.37
0.80
2.98
3.94
22.38
12.72
CATA_NLSVEDAAR
0.14
0.40
2.91
5.30
24.86
109.32
B
Q-TOF
C
Sample
A
Peptide
theor. fmol
140
900
120
TQ
OT
QTOF
21
OT
TQ
QTOF
6
OT
QTOF
60
Inter-Lab %CV
40
Sample A (0.8 fmol)
Intra-Lab %CV
CATA_FSTVAGESGSADTVR
NADPH_EGHLSPDIVAEQK
7
80
19
Sample A (4 fmol)
CAH1_GGPFSDSYR
22
Sample A
100
22
20
TQ
Sample A (20 fmol)
23
120
23
0.7
20
0.6
D
E
A
B
C
Average fmol measured
CAH1_GGPFSDSYR
160
D
0.5
E
CV(%)
20
40
100
200
20.44
40.20
102.97
200.38
1000
3.47
2.71
3.65
1.80
14.84
4.31
19.57
139.73
792.83
7.23
30.28
32.62
27.68
4.19
90.52
212.91
1182.04
21.38
9.12
31.40
0.71
11.31
8.00
20.00
40.00
200.00
NQO1_EGHLSPDIVAEQK
3.63
7.54
18.53
35.77
179.64
58.66
23.84
16.67
13.41
NQO1_ALIVLAHSER
1.68
3.56
8.28
18.56
981.40
89.12
3.27
3.00
3.97
CRP_ESDTSYVSLK
4.86
11.05
19.15
37.98
188.45
48.40
37.45
CRP_GYSIFSYATK
2.13
4.96
14.12
24.56
148.97
16.25
18.92
0.80
1.60
4.00
8.00
40.00
21
20
fmol measured
theor. fmol
25
24
180
950
fmol measured
CRP_GYSIFSYATK
220
200
1000
CATA_FSTVAGESGSADTVR
NADPH_EGHLSPDIVAEQK
1150
1100
1050
Sample E (40 fmol)
Sample E (200 fmol)
CAH1_GGPFSDSYR
1.37
986.23
934.06
29.72
theor. fmol
0
E
3.48
181.99
124.25
15.13
NADPH_EGHLSPDIVAEQK
Intra-Lab %CV
5
%CV
A
1.84
CV(%)
200
198.71
86.40
73.73
CAH2_SADFTNFDPR
fmol measured
E
100
101.79
39.73
29.31
fmol measured
D
Average fmol measured
40
39.82
18.22
19.66
fmol measured
A
20
20.01
CAH1_VLDALQAIK
CAH2_AVQQPDGLAVLGIFLK
fmol measured
Sample
Peptide
Inter-Lab %CV
9.00
0.51
0.72
theor. fmol
15
10
7.42
PPIA_FEDENFILK
PPIA_VSFELFADK
CAH1_GGPFSDSYR
20
%CV
Sample
Peptide
theor. fmol
CAH1_GGPFSDSYR
5
4
0
0.4
0.3
0.2
CAH1_VLDALQAIK
21.19
41.07
95.07
204.44
CAH2_AVQQPDGLAVLGIFLK
19.40
43.28
107.15
CAH2_SADFTNFDPR
20.71
43.44
4.00
theor. fmol
theor. fmol
977.43
PPIA_FEDENFILK
0.51
1.14
3.16
4.65
24.84
PPIA_VSFELFADK
0.42
0.94
3.32
4.26
22.47
CATA_GAGAFGYFEVTHDITK
2.70
1.10
2.25
5.01
CATA_FSTVAGESGSADTVR
0.28
1.16
3.54
5.02
22.84
CATA_NLSVEDAAR
0.28
0.75
1.81
4.39
31.48
19
0.1
15.39
24.77
39.59
93.92
49.89
5.60
7.70
21.92
7.51
0.20
20.08
y = 0.9987x
R² = 1
600
400
OT
QTOF
TQ
OT
QTOF
TQ B
QTOF B
OT B
0.96
0.94
QTOF E
OT E
1
0.9
0.8
0.4
0.2
TQ E
0.2
CAH2_AVQQPDGLAVLGIFLK
1
0.8
0.6
0.4
10
Solid bars represent the measured % coefficients of variation (%CV)
between the three replicate analysis performed, averaged for all datasets.
Each bar is the %CV value for the measurement of the indicated peptide.
Error bars indicate the standard deviation of the values for all labs. Bars
are colored according to the concentration tiers in the samples. Empty
bars represent the value for Inter-Laboratory %CV for the corresponding
peptide measurement. The graphs for the most concentrated sample (E)
and the most diluted (A) are shown.
1.2
CAH1_VLDALQAI[K]
0.98
0.6
100
1.02
1
0.8
200
0
CAH2_AVQQPDGLAVLGIFL[K]
0.7
TQ E
0.6
QTOF E
0.5
OT E
0.4
0.3
0.2
0.92
0
0
1
0
200
400
600
theoretical fmol
800
1000
1
10
100
1000
0.9
10000
log theoretical fmol
NADPH
1.2
NADPH
1000
200
NADPH_EGHLSPDIVAEQK
NADPH_ALIVLAHSER
160
y = 0.8673x
R² = 0.9998
120
80
y = 0.4689x
R² = 0.9999
40
0.8
80
120
theoretical fmol
160
0.95
100
OT B
0.9
0.4
10
PPIA
100
log theoretical fmol
TQ E
QTOF E
0.85
0
1
200
TQ B
QTOF B
0.6
10
1
40
CATA_FSTVAGESGSADTVR
1
0.2
0
0
CATA_FSTVAGESGSADTVR
1
NADPH_EGHLSPDIVAEQK
NADPH_ALIVLAHSER
log measured fmol
measured fmol
CAH1_VLDALQAIK
1
1000
log measured fmol
measured fmol
1.2
CAH1_GGPFSDSYR
CAH1_VLDALQAIK
y = 1.0153x
R² = 1
TQ
REPRODUCIBILITY OF TRANSITION PATTERN
CAH1
10000
800
QTOF
Representative boxplots of the distribution of absolute concentration
measurements for a peptide of each of the concentration tiers. The graphs for
the most concentrated sample (E) and the most diluted (A) are shown.
Measurements are grouped by type of measurement/instrument: TQ: SRM on
triple quadrupole; OT, QTOF: pseudoSRM in orbitrap and Q-TOF instruments,
respectively. In general, no major differences in terms of median value or
dispersion were observed between the three groups.
25.76
14.23
LINEARITY OF THE RESPONSE
CAH1_GGPFSDSYR
CAH1_VLDALQAIK
1000
OT
3.18
7.97
97.18
0
2
TQ
CAH1
1200
3
18
6.86
3.04
Tables summarizing the measurements of absolute concentration for each peptide and sample,
averaged for each group of instruments, as indicated, and across all 30 datasets. The %CV of
variation of the measurements are shown on the right. Red scale highlights %CV values above 20%.
0.8
1000
OT E
Bar plots (left panels) represent the contribution of each of the three monitored transitions
to the total MS signal, for three example peptides, for each instrument/laboratory. To
compare the patterns, the dotproducts of the normalized vectors defined by the three
contributions and the one corresponding to the average of all laboratories have been
calculated. The plots on the right panels show the dotproduct values for each lab, grouped
by type of instrument.
The two peptide graphs shown on the left side are representative of the behaviour of most
of the signals, showing no major differences between instruments, irrespective of the signal
level (dotproducts for samples B and E shown for comparison).
The plots shown on top right are an example of a peptide for which different labs selected
different patterns, probably due to the presence of interfering signals. Two different patterns
are clearly seen both in he bar or dotproduct plots, not depending on the instrument type.
Quantification for this peptide shows accordingly a higher variability between laboratories.
PPIA
30
100
PPIA_FEDENFILK
PPIA_VSFELFADK
PPIA_FEDENFILK
PPIA_VSFELFADK
25
y = 0.5552x
R² = 0.9999
20
15
log measured fmol
measured fmol
EUPA 2013
In order to evaluate the robustness and reproducibility, within and across laboratories,
of the SRM and pseudo-SRM quantification methodology, we set up a multi-centric
study (PME8) carried out at 27 laboratories, including ProteoRed-ISCIII network of
proteomics facilities in Spain, several EuPA members, and other laboratories
worldwide.
Each participant laboratory received a set of 5 different samples A-E, prepared by
spiking different amounts of the Sigma-Aldrich MSQC1 standard into a yeast lysate
digest.
The five samples were analyzed in triplicate by SRM or pseudo-SRM using similar
chromatographic and spectrometric conditions at the different laboratories and with
different instruments. Each laboratory reported results on relative quantification (fold
changes between A-E samples) and absolute quantification based on the labeled
peptide standards
y = 0.5579x
R² = 0.9993
10
10
CONCLUSIONS
1
0.1
1
10
100
The results demonstrate a good degree of reproducibility of targeted quantification measurements by SRM at different laboratories, irrespective of the
method of analysis and the spectrometer used.
5
0
0
0
5
10
15
20
25
30
theoretical fmol
35
40
45
log theoretical fmol
Plots of measured amounts (average of 30 datasets) in fmol versus theoretical amounts, in
each of the five samples, for two different peptides of a protein of each concentration tier.
Error bars represent the standard deviation of the measurements across labs. Graphs on the
right show the linearity in log-log scale. A good linearity of the response was observed for all
peptides in the measured ranges.
A deviation from the expected theoretical amounts was observed in some cases. In the case
of NADPH peptides (middle graph), both peptides give a different quantification, being one of
them around half of the theoretical amount. This could be the result of an uncomplete
digestion of the protein during sample preparation. In the case of PPIA (bottom graph), both
peptides give the same quantification, but the value is about half of the theoretical amount.
This could potentially be due to unaccuracy in the quantification of the protein standard.
 The average Inter-Laboratory %CV of the measured absolute protein amounts ranges from less than 10%CV for Tier 1 proteins, to 40-60% for the
proteins at the lowest concentrations.
 The pattern of relative intensities of the transitions monitored is fairly consistent among different instruments and fragmentation modes,
underscoring the utility of spectral databases for the design of quantification methods.
The results obtained at each laboratory allow the assessment of the limitations in sensitivity and limits of quantification under the diverse analytical
conditions used
CONCLUSIONS
The results demonstrate a good degree of reproducibility of targeted
quantification measurements by SRM at different laboratories,
irrespective of the method of analysis and the spectrometer used.
 The average Inter-Laboratory %CV of the measured absolute
protein amounts ranges from less than 10%CV for Tier 1 proteins, to
40-60% for the proteins at the lowest concentrations.
 The pattern of relative intensities of the transitions monitored is
fairly consistent among different instruments and fragmentation
modes, underscoring the utility of spectral databases for the design of
quantification methods.
The results obtained at each laboratory allow the assessment of the
limitations in sensitivity and limits of quantification under the diverse
analytical conditions used