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

P20

RE

P21

RE

P22

RE

P23

RE

P24

RE

P25

RE

P26

RE

P27

RE

P28

RE

P29

RE

P30

Replicate

48

50

52

54

56

58

60

62

Mea

s ure

d Ti

me

y9 - 1059.5065+ y8 - 930.4639+ y7 - 801.4213+ y5 - 645.3315+

50 52 54 56 58 60 62

Retention Time

010203040506070

Inte

nsity

(10^

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

nsity

(10^

3)

49.9

61.460.0

58.6

REP20 REP21 REP22 REP23 REP24 REP25 REP26 REP27 REP28 REP29 REP30Replicate

0

20

40

60

80

100

Peak

Are

a Pe

rcen

tage

y9 - 1059.5065+ y8 - 930.4639+ y7 - 801.4213+ y5 - 645.3315+

Finding Issues at a Glance (interference)HGFLPR

Unlabeled

Labeled13C R

A1_

01

A1_

02

A1_

03

A1_

04

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_ 0

1

F_ 0

2

F_ 0

3

F_ 0

4

0

20

40

60

80

100

P eak

Ar e

a P e

r cen

t ag e

y5 - 589.3457+ y4 - 532.3242+ y3 - 385.2558+

A1_

01

A1_

02

A1_

03

A1_

04

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_ 0

1

F_ 0

2

F_ 0

3

F_ 0

4

Replicate

0

20

40

60

80

100

P eak

Ar e

a P e

r cen

t ag e

Abbatiello, S.E. et al. Clin. Chem. 2010.

Finding Issues Early (lack of precision)Peptide area mean of 10 replicates

TAA

(15 .

4)

GF C

(16

.0)

DG

G (

17. 1

)

VLD

(19

.0)

CAV

(20

.8)

LVN

(21

.1)

DD

G (

24.3

)

VGP

(33.

6)

FFV

(35.

5)

Peptide

0

10

20

30

40

50

Peak

Are

a (1

0^6)

Peptide area mean of 10 replicates

New

TAA

(15 .

9)

GF C

(16

.3)

DG

G (

17.3

)

VLD

(18

.8)

CAV

(20

.0)

LVN

(20

.4)

DD

G (

22.8

)

VGP

(33.

2)

FFV

(34.

8)

Peptide

0

10

20

30

40

50

Peak

Are

a ( 1

0^6)

Peptide area CV of 10 replicates

TAA

(15.

4)

GFC

(16

.0)

DG

G (

17.1

)

VLD

(19

.0)

CAV

(20

.8)

LVN

(21

.1)

DD

G (

24.3

)

VGP

(33.

6)

FFV

(35.

5)

Peptide

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Peak

Are

a CV

Peptide area CV of 10 replicates

New

TAA

(15 .

9)

GF C

(16

.3)

DG

G (

17.3

)

VLD

(18

.8)

CAV

(20

.0)

LVN

(20

.4)

DD

G (

22.8

)

VGP

(33.

2)

FFV

(34.

8)

Peptide

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Peak

Are

a CV

Sharing Reports

Analysis of Study 9S reports with RDownstream 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

FWHM

pea

k wi

dth

in m

in

(max

imum

for 5

tran

sitio

ns)

Replicate Number

0.1

0.2

0.3

TAA1 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 usersJun-09Jul-09

Aug-09Sep-09Oct-09

Nov-09Dec-09Jan-10Feb-10

Mar-10

Apr-10

May-10Jun-10Jul-10

Aug-10Sep-10Oct-10

Nov-10Dec-10Jan-11Feb-11

Mar-11

Apr-11

May-11Jun-11Jul-11

Aug-11Sep-11Oct-11

Nov-11Dec-11Jan-12Feb-12

Mar-12

Apr-12

May-12Jun-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

xx

xx

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

Peak

Are

a (1

0^6)

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

Peak

Are

a (1

0^6)

y11 - 1187.6630+ y10 - 1088.5946+ y8 - 900.5149+b3 - 325.1870+ b4 - 424.2554+ b5 - 511.2875+

Bruker DIA Data (Precursor Ions)

idotp0.99idotp0.99idotp1.00

idotp1.00idotp0.99

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

Peak

Are

a (1

0^6)

precursor - 756.4250++ precursor [M+1] - 756.9265++precursor [M+2] - 757.4278++

idotp0.99idotp0.99idotp1.00

idotp1.00idotp0.99

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

Peak

Are

a (1

0^6)

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