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