Targeted Proteomics
Environment
Quantitative Proteomics with Bruker Q-TOF
Instruments and Skyline
Brendan MacLean
Quantitative Proteomics
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Spectrum-based
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Spectral counting
Isobaric tags
Chromatography-based
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SRM
MS1 chromatogram extraction
Targeted MS/MS
Data independent acquisition (DIA)
Quantitative Proteomics
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Spectrum-based
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Spectral counting
Isobaric tags
Chromatography-based
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SRM
MS1 chromatogram extraction
Targeted MS/MS
Data independent acquisition (DIA)
micrOTOF-Q and maXis series
2010 Multi-Instrument SRM Tool for
NCI CPTAC Verification Working Group
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AB SCIEX
4000 Q Trap
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ThermoScientific
TSQ Ultra
Kessner, D. et al. Bioinformatics. 2008.
Support Multiple Instrument Vendors
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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
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Global Proteome Machine
MacCoss Lab
NIST
Peptide Atlas
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Build your own from peptide search results
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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
Finding Issue Quickly (wrong peak)
IVGYLDEEGVLDQNR
Peak Area Percentage
y9 - 1059.5065+
y8 - 930.4639+
y7 - 801.4213+
y5 - 645.3315+
100
80
60
40
20
0
REP20
REP21
REP22
REP23
REP24
REP25
REP26
REP27
REP28
REP29
REP30
Replicate
y9 - 1059.5065+
y8 - 930.4639+
y7 - 801.4213+
y5 - 645.3315+
62
58
56
54
52
50
Replicate
REP30
REP29
REP28
REP27
REP26
REP25
REP24
REP23
REP22
REP21
48
REP20
Measured Time
60
Labeled
13C R
B_ 04
C_ 01
C_ 02
C_ 03
B_ 04
C_ 01
C_ 02
C_ 03
D_ 02
D_ 03
D_ 04
E_ 01
E_ 02
E_ 03
E_ 04
F_ 01
F_ 02
F_ 03
F_ 04
D_ 02
D_ 03
D_ 04
E_ 01
E_ 02
E_ 03
E_ 04
F_ 01
F_ 02
F_ 03
F_ 04
D_ 01
B_ 03
B_ 03
y4 - 532.3242+
D_ 01
B_ 02
B_ 02
y5 - 589.3457+
C_ 04
B_ 01
B_ 01
HGFLPR
C_ 04
A1_ 04
A1_ 04
0
A1_ 03
20
A1_ 03
40
A1_ 02
60
A1_ 02
80
A1_ 01
100
A1_ 01
Peak Area Percentage
Unlabeled
Peak Area Percentage
Finding Issues at a Glance (interference)
y3 - 385.2558+
100
80
60
40
20
0
Replicate
Abbatiello, S.E. et al. Clin. Chem. 2010.
Peptide
0.6
0.6
0.4
0.3
0.5
0.2
0.2
0.1
0.1
0.0
0.0
Peptide
FFV (34.8)
0.7
VGP (33.2)
Peptide area CV of 10 replicates
DDG (22.8)
0.7
LVN (20.4)
Peptide
CAV (20.0)
FFV (34.8)
VGP (33.2)
DDG (22.8)
LVN (20.4)
CAV (20.0)
0
VLD (18.8)
0
VLD (18.8)
10
DGG (17.3)
10
DGG (17.3)
40
GFC (16.3)
20
TAA (15.9)
30
Peak Area (10^6)
50
GFC (16.3)
0.5
Peak Area CV
FFV (35.5)
VGP (33.6)
DDG (24.3)
LVN (21.1)
CAV (20.8)
VLD (19.0)
DGG (17.1)
GFC (16.0)
Peptide area mean of 10 replicates
TAA (15.9)
FFV (35.5)
VGP (33.6)
DDG (24.3)
LVN (21.1)
CAV (20.8)
VLD (19.0)
DGG (17.1)
GFC (16.0)
TAA (15.4)
Peak Area (10^6)
50
TAA (15.4)
Peak Area CV
Finding Issues Early (lack of precision)
Peptide area mean of 10 replicates
40
30
New
20
Peptide
Peptide area CV of 10 replicates
New
0.4
0.3
Sharing
Reports
Downstream Analysis with Statistical Tools
Analysis of Study 9S reports with R
Peak Width (FWHM)
1 2 3 4 5 6 7 8 9 10
TAA
GFC
DGG
0 .3
0 .2
FWHM peak width in min
(maximum for 5 transitions)
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Site
0 .1
VLD
CAV
LVN
0.3
0.2
0.1
DDG
VGP
FFV
0 .3
0 .2
0 .1
1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
Replicate Number
1
2
3
5
5
5
5
6
7
8
8
9
9
9
0
2
2
4
6
6A
5
3
6
6A
5
8
!LC
!LC
!LC
!LC
Deeper Analysis with Custom Tools
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Analysis of reports with Retention Time Viewer (Java program)
http://www.buckinstitute.org/Labs/thegibson/resources.asp
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660 registered users
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Skyline Adoption (>12,500 Installations)
New Skyline Installations by Month
900
800
700
600
500
400
300
200
100
0
4500
Skyline Use
4000
3500
Skyline Instances Started Trailing 7 Days
3000
ASMS
2011
2500
ASMS
2012
Dev 64 Release
Dev Release
2000
Instances
1.3 64 Release
1500
ASMS
2010
1.3 Release
1.2 Release
1000
500
1.1 Release
Pre-1.1 Release
0
>100 citations
2012 Support Multiple Instrument Vendors
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Full-Scan
Exporting isolation lists & native methods
Importing native instrument output files
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AB SCIEX
Agilent Technologies
SWATH™
DIA
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Bruker
DIA & All Ions DIA
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Thermo-Scientific
Waters
DIA & Multiplexed DIA
MSe™
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Skyline MS1 Full-Scan Settings
Skyline MS1 Filtering Data
Schilling, B. et al. Mol. Cell. Prot. 2012.
Targeted MS/MS
Q1 / Trap
CID
Mass
Analyzer
Ion
Source
2 m/z isolation
MS/MS scans
Skyline Targeted MS/MS Settings
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 Scheme
SVEDFMAAMQR
Precursor m/z: 616
VGGNGADYALATK
Precursor m/z: 619
precursor m/z
600
610
10 m/z
616 620
630
640
619
x
x
x
x
Retention time
Venable, J.D. et al. Nat. Meth. 2004.
650
DIA Chromatogram Extraction
Q1 / Trap
CID
Mass
Analyzer
Ion
Source
10 m/z isolation
MS/MS scans
DIA Chromatogram Extraction Trade-Offs
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Sensitivity
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Dwell / Accumulation time
Cycle time
Selectivity lost by wide precursor isolation
Selectivity gained vs. SRM by narrow product extraction
MS/MS scan
Gillet, L.C. et al. Mol. Cell. Prot. 2012.
Skyline Bruker DIA Settings
(25 m/z Extraction Windows)
Gillet, L.C. et al. Mol. Cell. Prot. 2012.
Bruker DIA Data (Product Ions)
Bruker DIA Data (Precursor Ions)
Bruker All Ions DIA Settings
(50 – 1500 m/z Range)
Bruker All Ions DIA Data
Getting Started
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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)
MS1 Full-Scan Filtering
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Targeted MS/MS
Data Independent Acquisition
Support board and issues list
8 other tutorials & 3 instructional videos (full-scan video soon)