Targeted Proteomics Environment
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Transcript of Targeted Proteomics Environment
Targeted ProteomicsEnvironment
Quantitative Proteomics with Bruker Q-TOF Instruments and Skyline
Brendan MacLean
Quantitative Proteomics Spectrum-based
Spectral counting Isobaric tags
Chromatography-based SRM MS1 chromatogram extraction Targeted MS/MS Data independent acquisition (DIA)
Quantitative Proteomics Spectrum-based
Spectral counting Isobaric tags
Chromatography-based SRM MS1 chromatogram extraction Targeted MS/MS Data independent acquisition (DIA)
micrOTOF-Q and maXis series
2010 Multi-Instrument SRM Tool forNCI CPTAC Verification Working Group
AB SCIEX4000 Q Trap
Thermo-ScientificTSQ Ultra
Kessner, D. et al. Bioinformatics. 2008.
Support Multiple Instrument Vendors Selected Reaction Monitoring Exporting transition lists & native methods Importing native instrument output files
AB Sciex Agilent Technologies Thermo-Scientific Waters
Graphic Display of Information
MS/MS Spectral Library Sources Global Proteome Machine MacCoss Lab NIST Peptide Atlas
Build your own from peptide search results Mascot Myrimatch / IDPicker OMSSA Protein Pilot Protein Prospector Scaffold – mzIdentML / MGF Spectrum Mill TPP – pepXML / mzXML files – Peptide Atlas X! Tandem Waters MSe
Spectral Library Explorer
RE
P2
0
RE
P2
1
RE
P2
2
RE
P2
3
RE
P2
4
RE
P2
5
RE
P2
6
RE
P2
7
RE
P2
8
RE
P2
9
RE
P3
0
Replicate
48
50
52
54
56
58
60
62
Me
asu
red
Tim
e
y9 - 1059.5065+ y8 - 930.4639+ y7 - 801.4213+ y5 - 645.3315+
50 52 54 56 58 60 62
Retention Time
0
10
20
30
40
50
60
70
Inte
ns
ity
(1
0^
3)
58.8
50.4
62.561.7
60.6
Finding Issue Quickly (wrong peak)IVGYLDEEGVLDQNR
50 52 54 56 58 60
Retention Time
0
50
100
150
200
Inte
ns
ity
(1
0^
3)
49.9
61.460.0
58.6
REP20 REP21 REP22 REP23 REP24 REP25 REP26 REP27 REP28 REP29 REP30
Replicate
0
20
40
60
80
100
Pe
ak
Are
a P
e rc
enta
ge
y9 - 1059.5065+ y8 - 930.4639+ y7 - 801.4213+ y5 - 645.3315+
Finding Issues at a Glance (interference)HGFLPR
Unlabeled
Labeled13C R
A1
_ 0
1
A1
_ 0
2
A1
_ 0
3
A1
_ 0
4
B_
01
B_
02
B_
03
B_
04
C_
01
C_
02
C_
03
C_
04
D_
01
D_
02
D_
03
D_
04
E_
01
E_
02
E_
03
E_
04
F_
01
F_
02
F_
03
F_
04
0
20
40
60
80
100
Pe
ak A
r ea
Per
cen
t ag
e
y5 - 589.3457+ y4 - 532.3242+ y3 - 385.2558+
A1
_ 0
1
A1
_ 0
2
A1
_ 0
3
A1
_ 0
4
B_
01
B_
02
B_
03
B_
04
C_
01
C_
02
C_
03
C_
04
D_
01
D_
02
D_
03
D_
04
E_
01
E_
02
E_
03
E_
04
F_
01
F_
02
F_
03
F_
04
Replicate
0
20
40
60
80
100
Pe
ak A
r ea
Per
cen
t ag
e
Abbatiello, S.E. et al. Clin. Chem. 2010.
Finding Issues Early (lack of precision)Peptide area mean of 10 replicates
TA
A (
15 .
4 )
GF
C (
16 .
0 )
DG
G (
17 .
1 )
VL
D (
19 .
0 )
CA
V (
20 .
8 )
L VN
(2
1 .1 )
DD
G (
24 .
3 )
VG
P (
33 .
6 )
FF
V (
35 .
5 )
Peptide
0
10
20
30
40
50
Pe a
k A
rea
( 10^
6)
Peptide area mean of 10 replicates
New
T AA
(1
5 .9 )
GF
C (
16 .
3 )
DG
G (
17 .
3 )
VL
D (
18 .
8 )
CA
V (
20 .
0 )
L VN
(2
0 .4 )
DD
G (
22 .
8 )
VG
P (
33 .
2 )
FF
V (
34 .
8 )
Peptide
0
10
20
30
40
50
Pe a
k A
rea
( 10^
6)
Peptide area CV of 10 replicates
T AA
(1
5 .4 )
GF
C (
16 .
0 )
DG
G (
17 .
1 )
VL
D (
19 .
0 )
CA
V (
20 .
8 )
L VN
(2
1 .1 )
DD
G (
24 .
3 )
VG
P (
33 .
6 )
FF
V (
35 .
5 )
Peptide
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Pe a
k A
rea
CV
Peptide area CV of 10 replicates
New
TA
A (
15 .
9 )
GF
C (
16 .
3 )
DG
G (
17 .
3 )
VL
D (
18 .
8 )
CA
V (
20 .
0 )
L VN
(2
0 .4 )
DD
G (
22 .
8 )
VG
P (
33 .
2 )
FF
V (
34 .
8 )
Peptide
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Pe a
k A
rea
CV
Sharing Reports
Analysis of Study 9S reports with R
Downstream Analysis with Statistical Tools
Site1 92 03 25 25 45 65 6 A6 57 38 68 6 A9 59 8
! L C! L C
! L C! L C
FWH
M p
eak w
idth
in m
in
(maxim
um
for
5 t
ransi
tion
s)
Replicate Number
0.1
0.2
0.3
TAA
1 2 3 4 5 6 7 8 9 10
GFC DGG
VLD CAV
0.1
0.2
0.3
LVN
0.1
0.2
0.3
1 2 3 4 5 6 7 8 9 10
DDG VGP
1 2 3 4 5 6 7 8 9 10
FFV
Peak Width (FWHM)
Deeper Analysis with Custom Tools Analysis of reports with Retention Time Viewer (Java
program)
http://www.buckinstitute.org/Labs/thegibson/resources.asp
Skyline Adoption (>12,500 Installations)
660 registered users
Jun-09Jul-09
Aug-09
Sep-09
Oct-09
Nov-09
Dec-09Jan-10
Feb-10
Mar-10
Apr-10
May-10
Jun-10Jul-10
Aug-10
Sep-10
Oct-10
Nov-10
Dec-10Jan-11
Feb-11
Mar-11
Apr-11
May-11
Jun-11Jul-11
Aug-11
Sep-11
Oct-11
Nov-11
Dec-11Jan-12
Feb-12
Mar-12
Apr-12
May-12
Jun-12Jul-12
Aug-12
0
100
200
300
400
500
600
700
800
900
New Skyline Installations by Month
Skyline Use
Dev 64 Release
Dev Release
1.3 Release1.2 Release1.1 ReleasePre-1.1 Release
1.3 64 ReleaseASMS
2010
ASMS2011
Skyline Instances Started Trailing 7 Days
Inst
ance
s
3500
3000
2500
1500
1000
500
0
2000
4000
4500
>100 citations
ASMS2012
2012 Support Multiple Instrument Vendors Full-Scan Exporting isolation lists & native methods Importing native instrument output files
AB SCIEX SWATH™ Agilent Technologies DIA Bruker DIA & All Ions DIA Thermo-Scientific DIA & Multiplexed
DIA Waters MSe™
Skyline MS1 Full-Scan Settings
Tutorial
Skyline MS1 Filtering Data
Schilling, B. et al. Mol. Cell. Prot. 2012.
Targeted MS/MS
IonSource
CIDQ1 / TrapMass
Analyzer
MS/MS scans
time (scan #)
inte
nsit
y m/zm/z
m/zm/z
521.27
2 m/z isolation
Skyline Targeted MS/MS Settings
Tutorial
Skyline Targeted MS/MS
Sherrod, S. et al. J. Prot. Res. 2012.
Skyline Targeted MS/MS
Sherrod, S. et al. J. Prot. Res. 2012.
Traditional DIA Isolation Schemeprecursor m/z
Retention time
10 m/z
600 610 620 630 640 650SVEDFMAAMQRPrecursor m/z: 616 616
x
x
x
x
VGGNGADYALATKPrecursor m/z: 619
619
Venable, J.D. et al. Nat. Meth. 2004.
DIA Chromatogram Extraction
IonSource
CIDQ1 / TrapMass
Analyzer
MS/MS scans
time (scan #)
inte
nsit
y m/zm/z
m/zm/z
10 m/z isolation
DIA Chromatogram Extraction Trade-Offs Sensitivity
Dwell / Accumulation time Cycle time
Selectivity lost by wide precursor isolation Selectivity gained vs. SRM by narrow product
extraction
Gillet, L.C. et al. Mol. Cell. Prot. 2012.
MS/MS scan
Skyline Bruker DIA Settings(25 m/z Extraction Windows)
Gillet, L.C. et al. Mol. Cell. Prot. 2012.
Bruker DIA Data (Product Ions)
dotp0.87
dotp0.87dotp0.88
dotp0.87
dotp0.87
dotp0.87dotp0.87dotp0.87dotp0.88
dotp0.87
Libr
ary
25pm
ol_1
918
25pm
ol_1
923
25pm
ol_1
926
25pm
ol_1
930
25pm
ol_1
932
5pm
ol_1
919
5pm
ol_1
922
5pm
ol_1
927
5pm
ol_1
929
5pm
ol_1
933
Replicate
0
10
20
30
Pe
ak
Are
a (
10
^6
)
y11 - 1187.6630+ y10 - 1088.5946+ y8 - 900.5149+b3 - 325.1870+ b4 - 424.2554+ b5 - 511.2875+
dotp0.87
dotp0.87dotp0.88
dotp0.87
dotp0.87
dotp0.87dotp0.87dotp0.87dotp0.88
dotp0.87
Libr
ary
25pm
ol_1
918
25pm
ol_1
923
25pm
ol_1
926
25pm
ol_1
930
25pm
ol_1
932
5pm
ol_1
919
5pm
ol_1
922
5pm
ol_1
927
5pm
ol_1
929
5pm
ol_1
933
Replicate
0
10
20
30
Pe
ak
Are
a (
10
^6
)
y11 - 1187.6630+ y10 - 1088.5946+ y8 - 900.5149+b3 - 325.1870+ b4 - 424.2554+ b5 - 511.2875+
Bruker DIA Data (Precursor Ions)
idotp0.99
idotp0.99idotp1.00
idotp1.00
idotp0.99
idotp0.99idotp0.99idotp0.98idotp0.99idotp0.99
Exp
ecte
d
25pm
ol_1
918
25pm
ol_1
923
25pm
ol_1
926
25pm
ol_1
930
25pm
ol_1
932
5pm
ol_1
919
5pm
ol_1
922
5pm
ol_1
927
5pm
ol_1
929
5pm
ol_1
933
Replicate
0
2
4
6
8
10
12
14
16
Pe
ak
Are
a (
10
^6
)
precursor - 756.4250++ precursor [M+1] - 756.9265++precursor [M+2] - 757.4278++
idotp0.99
idotp0.99idotp1.00
idotp1.00
idotp0.99
idotp0.99idotp0.99idotp0.98idotp0.99idotp0.99
Exp
ecte
d
25pm
ol_1
918
25pm
ol_1
923
25pm
ol_1
926
25pm
ol_1
930
25pm
ol_1
932
5pm
ol_1
919
5pm
ol_1
922
5pm
ol_1
927
5pm
ol_1
929
5pm
ol_1
933
Replicate
0
2
4
6
8
10
12
14
16
Pe
ak
Are
a (
10
^6
)
precursor - 756.4250++ precursor [M+1] - 756.9265++precursor [M+2] - 757.4278++
Bruker All Ions DIA Settings(50 – 1500 m/z Range)
Bruker All Ions DIA Data
Getting Started Freely available & Open Source
http://proteome.gs.washington.edu/software/skyline Self-updating web installation (v1.4 coming soon) 2 full-scan filtering tutorials (DIA coming soon)
Support board and issues list 8 other tutorials & 3 instructional videos (full-scan
video soon)
MS1 Full-Scan Filtering Targeted MS/MS Data Independent Acquisition
Coming Soon