Targeted Proteomics Environment

29
Targeted Proteomics Environment Status of the Skyline open-source software project five years after its inception Brendan MacLean

description

Targeted Proteomics Environment. Status of the Skyline open-source software project five years after its inception. Brendan MacLean. User Community After 4 Years. 560 registered users 150 registered for this meeting 4 new papers in May - PowerPoint PPT Presentation

Transcript of Targeted Proteomics Environment

Page 1: Targeted Proteomics Environment

Targeted ProteomicsEnvironment

Status of the Skyline open-source software project five years after its inception

Brendan MacLean

Page 2: Targeted Proteomics Environment

User Community After 4 Years 560 registered users 150 registered for this meeting 4 new papers in May

Platform Independent and Label-free Quantitation of Proteomic Data using MS1 Extracted Ion Chromatograms in Skyline – Mol. Cel. Prot.

Label-Free Quantitation of Protein Modifications by Pseudo-Selected Reaction Monitoring with Internal Reference Peptides – J. Prot. Res.

Using iRT, a Normalized Retention Time for More Targeted Measurement of Peptides - Proteomics

The Development of Selected Reaction Monitoring Methods for Targeted Proteomics via Empirical Refinement - Proteomics

25 abstracts at ASMS mention Skyline 75 citations of original paper (after 2 ½ years)

30 in 2012

Page 3: Targeted Proteomics Environment

User Community After 5 Years 1000+ registered users 250 registered for this meeting More papers and press

Targeted proteomics Nature Methods Method of the Year in 2012 MS1 Label-free Quantification Using Ion Intensity Chromatograms in

Skyline (Research and Clinical Applications) (book chapter) Design, Implementation, and Multi-Site Evaluation of a System

Suitability Protocol for the Quantitative Assessment of Instrument Performance in LC-MRM-MS - MCP

Viewing the Targeted Proteomics Horizon with Skyline – eProtein feature

37 abstracts at ASMS mention Skyline 200+ citations of original paper

Page 4: Targeted Proteomics Environment

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

ASMS2012

5000

Panorama2013

Page 5: Targeted Proteomics Environment

Skyline Web Site Visits (past 3 months)

Page 6: Targeted Proteomics Environment

Learning More Au PS Workshop, Melbourne (February) US HUPO Workshop, Baltimore (March) Skyline User Group Meeting! here (now) SRM Course, Zurich (July) Proteomics Course, Cold Springs Harbor (July)

Targeted Quant. Course, Seattle

September 9-14

Page 7: Targeted Proteomics Environment

Instrument Vendor Partnerships

Page 8: Targeted Proteomics Environment

Panorama Partnership Program 2 – 4 labs Seeking leading edge targeted proteomics

infrastructure Local Panorama server installation Direct collaboration with Skyline/Panorama

team Full support for 1 year

Page 9: Targeted Proteomics Environment

Prior Knowledge and Consistency Powerful enough to be used cross-lab / cross

experiment More powerful run-to-run

Relative ion abundance Spectral and chromatogram libraries

Retention time iRT

Does ensuring comparable measurements require ID?

Page 10: Targeted Proteomics Environment

Haptoglobin Li

brar

y

D_1

02_R

EP

1

D_1

02_R

EP

2

D_1

02_R

EP

3

D_1

03_R

EP

1

D_1

03_R

EP

2

D_1

03_R

EP

3

D_1

08_R

EP

1

D_1

08_R

EP

2

D_1

08_R

EP

3

D_1

38_R

EP

1

D_1

38_R

EP

2

D_1

38_R

EP

3

D_1

54_R

EP

1

D_1

54_R

EP

2

D_1

54_R

EP

3

D_1

72_R

EP

1

D_1

72_R

EP

2

D_1

72_R

EP

3

D_1

96_R

EP

1

D_1

96_R

EP

2

D_1

96_R

EP

3

H_1

46_R

EP

1

H_1

46_R

EP

2

H_1

46_R

EP

3

H_1

47_R

EP

1

H_1

47_R

EP

2

H_1

47_R

EP

3

H_1

48_R

EP

1

H_1

48_R

EP

2

H_1

48_R

EP

3

H_1

59_R

EP

1

H_1

59_R

EP

2

H_1

59_R

EP

3

H_1

60_R

EP

1

H_1

60_R

EP

2

H_1

60_R

EP

3

H_1

61_R

EP

1

H_1

61_R

EP

2

H_1

61_R

EP

3

H_1

62_R

EP

1

H_1

62_R

EP

2

H_1

62_R

EP

3

Replicate

0

20

40

60

80

100

Peak

Are

a Pe

rcen

tage

y13 - 1426.6696+ y9 - 1024.5310+ y7 - 854.4254+ y6 - 691.3621+y4 - 477.2304+ y3 - 363.1874+

Libr

ary

D_1

02_R

EP

1

D_1

02_R

EP

2

D_1

02_R

EP

3

D_1

03_R

EP

1

D_1

03_R

EP

2

D_1

03_R

EP

3

D_1

08_R

EP

1

D_1

08_R

EP

2

D_1

08_R

EP

3

D_1

38_R

EP

1

D_1

38_R

EP

2

D_1

38_R

EP

3

D_1

54_R

EP

1

D_1

54_R

EP

2

D_1

54_R

EP

3

D_1

72_R

EP

1

D_1

72_R

EP

2

D_1

72_R

EP

3

D_1

96_R

EP

1

D_1

96_R

EP

2

D_1

96_R

EP

3

H_1

46_R

EP

1

H_1

46_R

EP

2

H_1

46_R

EP

3

H_1

47_R

EP

1

H_1

47_R

EP

2

H_1

47_R

EP

3

H_1

48_R

EP

1

H_1

48_R

EP

2

H_1

48_R

EP

3

H_1

59_R

EP

1

H_1

59_R

EP

2

H_1

59_R

EP

3

H_1

60_R

EP

1

H_1

60_R

EP

2

H_1

60_R

EP

3

H_1

61_R

EP

1

H_1

61_R

EP

2

H_1

61_R

EP

3

H_1

62_R

EP

1

H_1

62_R

EP

2

H_1

62_R

EP

3

Replicate

0

20

40

60

80

100

Peak

Are

a Pe

rcen

tage

y13 - 1426.6696+ y9 - 1024.5310+ y7 - 854.4254+ y6 - 691.3621+y4 - 477.2304+ y3 - 363.1874+

D_1

02_R

EP

1

D_1

02_R

EP

2

D_1

02_R

EP

3

D_1

03_R

EP

1

D_1

03_R

EP

2

D_1

03_R

EP

3

D_1

08_R

EP

1

D_1

08_R

EP

2

D_1

08_R

EP

3

D_1

38_R

EP

1

D_1

38_R

EP

2

D_1

38_R

EP

3

D_1

54_R

EP

1

D_1

54_R

EP

2

D_1

54_R

EP

3

D_1

72_R

EP

1

D_1

72_R

EP

2

D_1

72_R

EP

3

D_1

96_R

EP

1

D_1

96_R

EP

2

D_1

96_R

EP

3

H_1

46_R

EP

1

H_1

46_R

EP

2

H_1

46_R

EP

3

H_1

47_R

EP

1

H_1

47_R

EP

2

H_1

47_R

EP

3

H_1

48_R

EP

1

H_1

48_R

EP

2

H_1

48_R

EP

3

H_1

59_R

EP

1

H_1

59_R

EP

2

H_1

59_R

EP

3

H_1

60_R

EP

1

H_1

60_R

EP

2

H_1

60_R

EP

3

H_1

61_R

EP

1

H_1

61_R

EP

2

H_1

61_R

EP

3

H_1

62_R

EP

1

H_1

62_R

EP

2

H_1

62_R

EP

3

Replicate

16.5

17.0

17.5

18.0

Ret

entio

n Ti

me

y13 - 1426.6696+ y9 - 1024.5310+ y7 - 854.4254+ y6 - 691.3621+y4 - 477.2304+ y3 - 363.1874+

D_1

02_R

EP

1

D_1

02_R

EP

2

D_1

02_R

EP

3

D_1

03_R

EP

1

D_1

03_R

EP

2

D_1

03_R

EP

3

D_1

08_R

EP

1

D_1

08_R

EP

2

D_1

08_R

EP

3

D_1

38_R

EP

1

D_1

38_R

EP

2

D_1

38_R

EP

3

D_1

54_R

EP

1

D_1

54_R

EP

2

D_1

54_R

EP

3

D_1

72_R

EP

1

D_1

72_R

EP

2

D_1

72_R

EP

3

D_1

96_R

EP

1

D_1

96_R

EP

2

D_1

96_R

EP

3

H_1

46_R

EP

1

H_1

46_R

EP

2

H_1

46_R

EP

3

H_1

47_R

EP

1

H_1

47_R

EP

2

H_1

47_R

EP

3

H_1

48_R

EP

1

H_1

48_R

EP

2

H_1

48_R

EP

3

H_1

59_R

EP

1

H_1

59_R

EP

2

H_1

59_R

EP

3

H_1

60_R

EP

1

H_1

60_R

EP

2

H_1

60_R

EP

3

H_1

61_R

EP

1

H_1

61_R

EP

2

H_1

61_R

EP

3

H_1

62_R

EP

1

H_1

62_R

EP

2

H_1

62_R

EP

3

Replicate

16.5

17.0

17.5

18.0

Ret

entio

n Ti

me

y13 - 1426.6696+ y9 - 1024.5310+ y7 - 854.4254+ y6 - 691.3621+y4 - 477.2304+ y3 - 363.1874+

LQTEGDGIYTLNSEK

Page 11: Targeted Proteomics Environment

Haptoglobin Li

brar

y

D_1

02_R

EP

1

D_1

02_R

EP

2

D_1

02_R

EP

3

D_1

03_R

EP

1

D_1

03_R

EP

2

D_1

03_R

EP

3

D_1

08_R

EP

1

D_1

08_R

EP

2

D_1

08_R

EP

3

D_1

38_R

EP

1

D_1

38_R

EP

2

D_1

38_R

EP

3

D_1

54_R

EP

1

D_1

54_R

EP

2

D_1

54_R

EP

3

D_1

72_R

EP

1

D_1

72_R

EP

2

D_1

72_R

EP

3

D_1

96_R

EP

1

D_1

96_R

EP

2

D_1

96_R

EP

3

H_1

46_R

EP

1

H_1

46_R

EP

2

H_1

46_R

EP

3

H_1

47_R

EP

1

H_1

47_R

EP

2

H_1

47_R

EP

3

H_1

48_R

EP

1

H_1

48_R

EP

2

H_1

48_R

EP

3

H_1

59_R

EP

1

H_1

59_R

EP

2

H_1

59_R

EP

3

H_1

60_R

EP

1

H_1

60_R

EP

2

H_1

60_R

EP

3

H_1

61_R

EP

1

H_1

61_R

EP

2

H_1

61_R

EP

3

H_1

62_R

EP

1

H_1

62_R

EP

2

H_1

62_R

EP

3

Replicate

0

2

4

6

8

Peak

Are

a (1

0^6)

y13 - 1426.6696+ y9 - 1024.5310+ y7 - 854.4254+ y6 - 691.3621+ y4 - 477.2304+y3 - 363.1874+

Libr

ary

D_1

02_R

EP

1

D_1

02_R

EP

2

D_1

02_R

EP

3

D_1

03_R

EP

1

D_1

03_R

EP

2

D_1

03_R

EP

3

D_1

08_R

EP

1

D_1

08_R

EP

2

D_1

08_R

EP

3

D_1

38_R

EP

1

D_1

38_R

EP

2

D_1

38_R

EP

3

D_1

54_R

EP

1

D_1

54_R

EP

2

D_1

54_R

EP

3

D_1

72_R

EP

1

D_1

72_R

EP

2

D_1

72_R

EP

3

D_1

96_R

EP

1

D_1

96_R

EP

2

D_1

96_R

EP

3

H_1

46_R

EP

1

H_1

46_R

EP

2

H_1

46_R

EP

3

H_1

47_R

EP

1

H_1

47_R

EP

2

H_1

47_R

EP

3

H_1

48_R

EP

1

H_1

48_R

EP

2

H_1

48_R

EP

3

H_1

59_R

EP

1

H_1

59_R

EP

2

H_1

59_R

EP

3

H_1

60_R

EP

1

H_1

60_R

EP

2

H_1

60_R

EP

3

H_1

61_R

EP

1

H_1

61_R

EP

2

H_1

61_R

EP

3

H_1

62_R

EP

1

H_1

62_R

EP

2

H_1

62_R

EP

3

Replicate

0

2

4

6

8

Peak

Are

a (1

0^6)

y13 - 1426.6696+ y9 - 1024.5310+ y7 - 854.4254+ y6 - 691.3621+ y4 - 477.2304+y3 - 363.1874+

LQTEGDGIYTLNSEK Li

brar

y

D_1

02_R

EP

1

D_1

02_R

EP

2

D_1

02_R

EP

3

D_1

03_R

EP

1

D_1

03_R

EP

2

D_1

03_R

EP

3

D_1

08_R

EP

1

D_1

08_R

EP

2

D_1

08_R

EP

3

D_1

38_R

EP

1

D_1

38_R

EP

2

D_1

38_R

EP

3

D_1

54_R

EP

1

D_1

54_R

EP

2

D_1

54_R

EP

3

D_1

72_R

EP

1

D_1

72_R

EP

2

D_1

72_R

EP

3

D_1

96_R

EP

1

D_1

96_R

EP

2

D_1

96_R

EP

3

H_1

46_R

EP

1

H_1

46_R

EP

2

H_1

46_R

EP

3

H_1

47_R

EP

1

H_1

47_R

EP

2

H_1

47_R

EP

3

H_1

48_R

EP

1

H_1

48_R

EP

2

H_1

48_R

EP

3

H_1

59_R

EP

1

H_1

59_R

EP

2

H_1

59_R

EP

3

H_1

60_R

EP

1

H_1

60_R

EP

2

H_1

60_R

EP

3

H_1

61_R

EP

1

H_1

61_R

EP

2

H_1

61_R

EP

3

H_1

62_R

EP

1

H_1

62_R

EP

2

H_1

62_R

EP

3

Replicate

0.0

0.5

1.0

1.5

2.0

Peak

Are

a (1

0^6)

y7 - 757.4818+ y6 - 658.4134+ y5 - 543.3865+ y4 - 430.3024+

Libr

ary

D_1

02_R

EP

1

D_1

02_R

EP

2

D_1

02_R

EP

3

D_1

03_R

EP

1

D_1

03_R

EP

2

D_1

03_R

EP

3

D_1

08_R

EP

1

D_1

08_R

EP

2

D_1

08_R

EP

3

D_1

38_R

EP

1

D_1

38_R

EP

2

D_1

38_R

EP

3

D_1

54_R

EP

1

D_1

54_R

EP

2

D_1

54_R

EP

3

D_1

72_R

EP

1

D_1

72_R

EP

2

D_1

72_R

EP

3

D_1

96_R

EP

1

D_1

96_R

EP

2

D_1

96_R

EP

3

H_1

46_R

EP

1

H_1

46_R

EP

2

H_1

46_R

EP

3

H_1

47_R

EP

1

H_1

47_R

EP

2

H_1

47_R

EP

3

H_1

48_R

EP

1

H_1

48_R

EP

2

H_1

48_R

EP

3

H_1

59_R

EP

1

H_1

59_R

EP

2

H_1

59_R

EP

3

H_1

60_R

EP

1

H_1

60_R

EP

2

H_1

60_R

EP

3

H_1

61_R

EP

1

H_1

61_R

EP

2

H_1

61_R

EP

3

H_1

62_R

EP

1

H_1

62_R

EP

2

H_1

62_R

EP

3

Replicate

0.0

0.5

1.0

1.5

2.0

Peak

Are

a (1

0^6)

y7 - 757.4818+ y6 - 658.4134+ y5 - 543.3865+ y4 - 430.3024+

SVVDIGLIK

Page 12: Targeted Proteomics Environment

Mitochondrial 39S ribosomal protein L9D

_102

_RE

P1

D_1

02_R

EP

2

D_1

02_R

EP

3

D_1

03_R

EP

1

D_1

03_R

EP

2

D_1

03_R

EP

3

D_1

08_R

EP

1

D_1

08_R

EP

2

D_1

08_R

EP

3

D_1

38_R

EP

1

D_1

38_R

EP

2

D_1

38_R

EP

3

D_1

54_R

EP

1

D_1

54_R

EP

2

D_1

54_R

EP

3

D_1

72_R

EP

1

D_1

72_R

EP

2

D_1

72_R

EP

3

D_1

96_R

EP

1

D_1

96_R

EP

2

D_1

96_R

EP

3

H_1

46_R

EP

1

H_1

46_R

EP

2

H_1

46_R

EP

3

H_1

47_R

EP

1

H_1

47_R

EP

2

H_1

47_R

EP

3

H_1

48_R

EP

1

H_1

48_R

EP

2

H_1

48_R

EP

3

H_1

59_R

EP

1

H_1

59_R

EP

2

H_1

59_R

EP

3

H_1

60_R

EP

1

H_1

60_R

EP

2

H_1

60_R

EP

3

H_1

61_R

EP

1

H_1

61_R

EP

2

H_1

61_R

EP

3

H_1

62_R

EP

1

H_1

62_R

EP

2

H_1

62_R

EP

3

Replicate

0

20

40

60

80

100

Peak

Are

a Pe

rcen

tage

y8 - 930.4944+ y7 - 744.4151+ y6 - 673.3780+ y5 - 616.3566+ y4 - 545.3194+y3 - 474.2823+

D_1

02_R

EP

1

D_1

02_R

EP

2

D_1

02_R

EP

3

D_1

03_R

EP

1

D_1

03_R

EP

2

D_1

03_R

EP

3

D_1

08_R

EP

1

D_1

08_R

EP

2

D_1

08_R

EP

3

D_1

38_R

EP

1

D_1

38_R

EP

2

D_1

38_R

EP

3

D_1

54_R

EP

1

D_1

54_R

EP

2

D_1

54_R

EP

3

D_1

72_R

EP

1

D_1

72_R

EP

2

D_1

72_R

EP

3

D_1

96_R

EP

1

D_1

96_R

EP

2

D_1

96_R

EP

3

H_1

46_R

EP

1

H_1

46_R

EP

2

H_1

46_R

EP

3

H_1

47_R

EP

1

H_1

47_R

EP

2

H_1

47_R

EP

3

H_1

48_R

EP

1

H_1

48_R

EP

2

H_1

48_R

EP

3

H_1

59_R

EP

1

H_1

59_R

EP

2

H_1

59_R

EP

3

H_1

60_R

EP

1

H_1

60_R

EP

2

H_1

60_R

EP

3

H_1

61_R

EP

1

H_1

61_R

EP

2

H_1

61_R

EP

3

H_1

62_R

EP

1

H_1

62_R

EP

2

H_1

62_R

EP

3

Replicate

0

20

40

60

80

100

Peak

Are

a Pe

rcen

tage

y8 - 930.4944+ y7 - 744.4151+ y6 - 673.3780+ y5 - 616.3566+ y4 - 545.3194+y3 - 474.2823+

D_1

02_R

EP

1

D_1

02_R

EP

2

D_1

02_R

EP

3

D_1

03_R

EP

1

D_1

03_R

EP

2

D_1

03_R

EP

3

D_1

08_R

EP

1

D_1

08_R

EP

2

D_1

08_R

EP

3

D_1

38_R

EP

1

D_1

38_R

EP

2

D_1

38_R

EP

3

D_1

54_R

EP

1

D_1

54_R

EP

2

D_1

54_R

EP

3

D_1

72_R

EP

1

D_1

72_R

EP

2

D_1

72_R

EP

3

D_1

96_R

EP

1

D_1

96_R

EP

2

D_1

96_R

EP

3

H_1

46_R

EP

1

H_1

46_R

EP

2

H_1

46_R

EP

3

H_1

47_R

EP

1

H_1

47_R

EP

2

H_1

47_R

EP

3

H_1

48_R

EP

1

H_1

48_R

EP

2

H_1

48_R

EP

3

H_1

59_R

EP

1

H_1

59_R

EP

2

H_1

59_R

EP

3

H_1

60_R

EP

1

H_1

60_R

EP

2

H_1

60_R

EP

3

H_1

61_R

EP

1

H_1

61_R

EP

2

H_1

61_R

EP

3

H_1

62_R

EP

1

H_1

62_R

EP

2

H_1

62_R

EP

3

Replicate

19.520.020.521.021.522.022.5

Ret

entio

n Ti

me

y8 - 930.4944+ y7 - 744.4151+ y6 - 673.3780+ y5 - 616.3566+ y4 - 545.3194+y3 - 474.2823+

D_1

02_R

EP

1

D_1

02_R

EP

2

D_1

02_R

EP

3

D_1

03_R

EP

1

D_1

03_R

EP

2

D_1

03_R

EP

3

D_1

08_R

EP

1

D_1

08_R

EP

2

D_1

08_R

EP

3

D_1

38_R

EP

1

D_1

38_R

EP

2

D_1

38_R

EP

3

D_1

54_R

EP

1

D_1

54_R

EP

2

D_1

54_R

EP

3

D_1

72_R

EP

1

D_1

72_R

EP

2

D_1

72_R

EP

3

D_1

96_R

EP

1

D_1

96_R

EP

2

D_1

96_R

EP

3

H_1

46_R

EP

1

H_1

46_R

EP

2

H_1

46_R

EP

3

H_1

47_R

EP

1

H_1

47_R

EP

2

H_1

47_R

EP

3

H_1

48_R

EP

1

H_1

48_R

EP

2

H_1

48_R

EP

3

H_1

59_R

EP

1

H_1

59_R

EP

2

H_1

59_R

EP

3

H_1

60_R

EP

1

H_1

60_R

EP

2

H_1

60_R

EP

3

H_1

61_R

EP

1

H_1

61_R

EP

2

H_1

61_R

EP

3

H_1

62_R

EP

1

H_1

62_R

EP

2

H_1

62_R

EP

3

Replicate

19.520.020.521.021.522.022.5

Ret

entio

n Ti

me

y8 - 930.4944+ y7 - 744.4151+ y6 - 673.3780+ y5 - 616.3566+ y4 - 545.3194+y3 - 474.2823+

CSSLLWAGAAWLR

Page 13: Targeted Proteomics Environment

Mitochondrial 39S ribosomal protein L9D

_102

_RE

P1

D_1

02_R

EP

2

D_1

02_R

EP

3

D_1

03_R

EP

1

D_1

03_R

EP

2

D_1

03_R

EP

3

D_1

08_R

EP

1

D_1

08_R

EP

2

D_1

08_R

EP

3

D_1

38_R

EP

1

D_1

38_R

EP

2

D_1

38_R

EP

3

D_1

54_R

EP

1

D_1

54_R

EP

2

D_1

54_R

EP

3

D_1

72_R

EP

1

D_1

72_R

EP

2

D_1

72_R

EP

3

D_1

96_R

EP

1

D_1

96_R

EP

2

D_1

96_R

EP

3

H_1

46_R

EP

1

H_1

46_R

EP

2

H_1

46_R

EP

3

H_1

47_R

EP

1

H_1

47_R

EP

2

H_1

47_R

EP

3

H_1

48_R

EP

1

H_1

48_R

EP

2

H_1

48_R

EP

3

H_1

59_R

EP

1

H_1

59_R

EP

2

H_1

59_R

EP

3

H_1

60_R

EP

1

H_1

60_R

EP

2

H_1

60_R

EP

3

H_1

61_R

EP

1

H_1

61_R

EP

2

H_1

61_R

EP

3

H_1

62_R

EP

1

H_1

62_R

EP

2

H_1

62_R

EP

3

Replicate

020406080

100120140

Peak

Are

a (1

0^3)

y8 - 930.4944+ y7 - 744.4151+ y6 - 673.3780+ y5 - 616.3566+ y4 - 545.3194+y3 - 474.2823+

D_1

02_R

EP

1

D_1

02_R

EP

2

D_1

02_R

EP

3

D_1

03_R

EP

1

D_1

03_R

EP

2

D_1

03_R

EP

3

D_1

08_R

EP

1

D_1

08_R

EP

2

D_1

08_R

EP

3

D_1

38_R

EP

1

D_1

38_R

EP

2

D_1

38_R

EP

3

D_1

54_R

EP

1

D_1

54_R

EP

2

D_1

54_R

EP

3

D_1

72_R

EP

1

D_1

72_R

EP

2

D_1

72_R

EP

3

D_1

96_R

EP

1

D_1

96_R

EP

2

D_1

96_R

EP

3

H_1

46_R

EP

1

H_1

46_R

EP

2

H_1

46_R

EP

3

H_1

47_R

EP

1

H_1

47_R

EP

2

H_1

47_R

EP

3

H_1

48_R

EP

1

H_1

48_R

EP

2

H_1

48_R

EP

3

H_1

59_R

EP

1

H_1

59_R

EP

2

H_1

59_R

EP

3

H_1

60_R

EP

1

H_1

60_R

EP

2

H_1

60_R

EP

3

H_1

61_R

EP

1

H_1

61_R

EP

2

H_1

61_R

EP

3

H_1

62_R

EP

1

H_1

62_R

EP

2

H_1

62_R

EP

3

Replicate

020406080

100120140

Peak

Are

a (1

0^3)

y8 - 930.4944+ y7 - 744.4151+ y6 - 673.3780+ y5 - 616.3566+ y4 - 545.3194+y3 - 474.2823+

D_1

02_R

EP

1

D_1

02_R

EP

2

D_1

02_R

EP

3

D_1

03_R

EP

1

D_1

03_R

EP

2

D_1

03_R

EP

3

D_1

08_R

EP

1

D_1

08_R

EP

2

D_1

08_R

EP

3

D_1

38_R

EP

1

D_1

38_R

EP

2

D_1

38_R

EP

3

D_1

54_R

EP

1

D_1

54_R

EP

2

D_1

54_R

EP

3

D_1

72_R

EP

1

D_1

72_R

EP

2

D_1

72_R

EP

3

D_1

96_R

EP

1

D_1

96_R

EP

2

D_1

96_R

EP

3

H_1

46_R

EP

1

H_1

46_R

EP

2

H_1

46_R

EP

3

H_1

47_R

EP

1

H_1

47_R

EP

2

H_1

47_R

EP

3

H_1

48_R

EP

1

H_1

48_R

EP

2

H_1

48_R

EP

3

H_1

59_R

EP

1

H_1

59_R

EP

2

H_1

59_R

EP

3

H_1

60_R

EP

1

H_1

60_R

EP

2

H_1

60_R

EP

3

H_1

61_R

EP

1

H_1

61_R

EP

2

H_1

61_R

EP

3

H_1

62_R

EP

1

H_1

62_R

EP

2

H_1

62_R

EP

3

Replicate

0

200

400

600

800

Peak

Are

a (1

0^3)

y9 - 961.5942+ y8 - 862.5257+ y6 - 664.3889+ y5 - 593.3518+ y4 - 496.2990+y3 - 359.2401+

D_1

02_R

EP

1

D_1

02_R

EP

2

D_1

02_R

EP

3

D_1

03_R

EP

1

D_1

03_R

EP

2

D_1

03_R

EP

3

D_1

08_R

EP

1

D_1

08_R

EP

2

D_1

08_R

EP

3

D_1

38_R

EP

1

D_1

38_R

EP

2

D_1

38_R

EP

3

D_1

54_R

EP

1

D_1

54_R

EP

2

D_1

54_R

EP

3

D_1

72_R

EP

1

D_1

72_R

EP

2

D_1

72_R

EP

3

D_1

96_R

EP

1

D_1

96_R

EP

2

D_1

96_R

EP

3

H_1

46_R

EP

1

H_1

46_R

EP

2

H_1

46_R

EP

3

H_1

47_R

EP

1

H_1

47_R

EP

2

H_1

47_R

EP

3

H_1

48_R

EP

1

H_1

48_R

EP

2

H_1

48_R

EP

3

H_1

59_R

EP

1

H_1

59_R

EP

2

H_1

59_R

EP

3

H_1

60_R

EP

1

H_1

60_R

EP

2

H_1

60_R

EP

3

H_1

61_R

EP

1

H_1

61_R

EP

2

H_1

61_R

EP

3

H_1

62_R

EP

1

H_1

62_R

EP

2

H_1

62_R

EP

3

Replicate

0

200

400

600

800

Peak

Are

a (1

0^3)

y9 - 961.5942+ y8 - 862.5257+ y6 - 664.3889+ y5 - 593.3518+ y4 - 496.2990+y3 - 359.2401+

SVVDIGLIK

CSSLLWAGAAWLR

Page 14: Targeted Proteomics Environment

CSSLLWAGAAWLRH_159_REP1

20.0 20.5 21.0 21.5

Retention Time

0

1000

2000

3000

4000

5000

Inte

nsity

20.7

21.6

20.4 20.6 20.8 21.0 21.2 21.4

Retention Time

0

20

40

60

80

100

120

140

Inte

nsity

21.021.4

D_138_REP3

18 19 20 21 22 23 24 25

Retention Time

0100200300400500600700800900

Inte

nsity

21.0

22.5

23.119.4

22.8

20.0

19.6

D_1

02_R

EP

1

D_1

03_R

EP

1

D_1

08_R

EP

1

D_1

38_R

EP

1

D_1

54_R

EP

1

D_1

72_R

EP

1

D_1

96_R

EP

1

H_1

46_R

EP

1

H_1

47_R

EP

1

H_1

48_R

EP

1

H_1

59_R

EP

1

H_1

60_R

EP

1

H_1

61_R

EP

1

H_1

62_R

EP

1

D_1

02_R

EP

2

D_1

03_R

EP

2

D_1

08_R

EP

2

D_1

38_R

EP

2

D_1

54_R

EP

2

D_1

72_R

EP

2

D_1

96_R

EP

2

H_1

46_R

EP

2

H_1

47_R

EP

2

H_1

48_R

EP

2

H_1

59_R

EP

2

H_1

60_R

EP

2

H_1

61_R

EP

2

H_1

62_R

EP

2

D_1

02_R

EP

3

D_1

03_R

EP

3

D_1

08_R

EP

3

D_1

38_R

EP

3

D_1

54_R

EP

3

D_1

72_R

EP

3

D_1

96_R

EP

3

H_1

46_R

EP

3

H_1

47_R

EP

3

H_1

48_R

EP

3

H_1

59_R

EP

3

H_1

60_R

EP

3

H_1

61_R

EP

3

H_1

62_R

EP

3

Replicate

19.520.020.521.021.522.022.5

Ret

entio

n Ti

me

y8 - 930.4944+ y7 - 744.4151+ y6 - 673.3780+ y5 - 616.3566+ y4 - 545.3194+y3 - 474.2823+

D_1

02_R

EP

1

D_1

03_R

EP

1

D_1

08_R

EP

1

D_1

38_R

EP

1

D_1

54_R

EP

1

D_1

72_R

EP

1

D_1

96_R

EP

1

H_1

46_R

EP

1

H_1

47_R

EP

1

H_1

48_R

EP

1

H_1

59_R

EP

1

H_1

60_R

EP

1

H_1

61_R

EP

1

H_1

62_R

EP

1

D_1

02_R

EP

2

D_1

03_R

EP

2

D_1

08_R

EP

2

D_1

38_R

EP

2

D_1

54_R

EP

2

D_1

72_R

EP

2

D_1

96_R

EP

2

H_1

46_R

EP

2

H_1

47_R

EP

2

H_1

48_R

EP

2

H_1

59_R

EP

2

H_1

60_R

EP

2

H_1

61_R

EP

2

H_1

62_R

EP

2

D_1

02_R

EP

3

D_1

03_R

EP

3

D_1

08_R

EP

3

D_1

38_R

EP

3

D_1

54_R

EP

3

D_1

72_R

EP

3

D_1

96_R

EP

3

H_1

46_R

EP

3

H_1

47_R

EP

3

H_1

48_R

EP

3

H_1

59_R

EP

3

H_1

60_R

EP

3

H_1

61_R

EP

3

H_1

62_R

EP

3

Replicate

19.520.020.521.021.522.022.5

Ret

entio

n Ti

me

y8 - 930.4944+ y7 - 744.4151+ y6 - 673.3780+ y5 - 616.3566+ y4 - 545.3194+y3 - 474.2823+

Page 15: Targeted Proteomics Environment

Truncated and Missing PeaksTGTNLMDFLSR

Page 16: Targeted Proteomics Environment

Deciphering the UnexpectedA

1A

3A

4A

5A

6A

7A

8A

9A

10 A11

A12 B

1B

2B

3B

4B

5B

6B

7B

8B

10 B11

B12 C

1C

2C

3C

4C

5C

6C

7C

8C

9C

10C

11C

12 D1

D2

D4

D5

D6

D7

D8

D9

D10

D11

D12 E

1E

2E

3E

4E

5E

6E

7E

8E

9E

10 E11

E12 F1 F2 F3 F4 F5 F6 F7 F8 F9 F1

0F1

1F1

2G

1G

2G

3G

4G

5G

6G

7G

8G

9G

10G

11G

12

Replicate

19.2

19.4

19.6

19.8

20.0

20.2

20.4

20.6

20.8

21.0

Ret

entio

n Ti

me

YGQTIRPICLPCTEGTTR - 708.3506+++ YGQTIRPICLPCTEGTTR - 711.6867+++ (heavy)

A1

A3

A4

A5

A6

A7

A8

A9

A10 A11

A12 B

1B

2B

3B

4B

5B

6B

7B

8B

10 B11

B12 C

1C

2C

3C

4C

5C

6C

7C

8C

9C

10C

11C

12 D1

D2

D4

D5

D6

D7

D8

D9

D10

D11

D12 E

1E

2E

3E

4E

5E

6E

7E

8E

9E

10 E11

E12 F1 F2 F3 F4 F5 F6 F7 F8 F9 F1

0F1

1F1

2G

1G

2G

3G

4G

5G

6G

7G

8G

9G

10G

11G

12

Replicate

19.2

19.4

19.6

19.8

20.0

20.2

20.4

20.6

20.8

21.0

Ret

entio

n Ti

me

YGQTIRPICLPCTEGTTR - 708.3506+++ YGQTIRPICLPCTEGTTR - 711.6867+++ (heavy) YGQTIRPICLPCTEGTTR

17 to 22-Feb-2012 16 to 17-Mar-2012

Page 17: Targeted Proteomics Environment

Aligned by iRTA

1A

3A

4A

5A

6A

7A

8A

9A

10 A11

A12 B

1B

2B

3B

4B

5B

6B

7B

8B

10 B11

B12 C

1C

2C

3C

4C

5C

6C

7C

8C

9C

10C

11C

12 D1

D2

D4

D5

D6

D7

D8

D9

D10

D11

D12 E

1E

2E

3E

4E

5E

6E

7E

8E

9E

10 E11

E12 F1 F2 F3 F4 F5 F6 F7 F8 F9 F1

0F1

1F1

2G

1G

2G

3G

4G

5G

6G

7G

8G

9G

10G

11G

12

Replicate

37

38

39

40

41

42

43

iRT

(Hut

tenh

ain)

Sco

re

YGQTIRPICLPCTEGTTR - 708.3506+++ YGQTIRPICLPCTEGTTR - 711.6867+++ (heavy)

A1

A3

A4

A5

A6

A7

A8

A9

A10 A11

A12 B

1B

2B

3B

4B

5B

6B

7B

8B

10 B11

B12 C

1C

2C

3C

4C

5C

6C

7C

8C

9C

10C

11C

12 D1

D2

D4

D5

D6

D7

D8

D9

D10

D11

D12 E

1E

2E

3E

4E

5E

6E

7E

8E

9E

10 E11

E12 F1 F2 F3 F4 F5 F6 F7 F8 F9 F1

0F1

1F1

2G

1G

2G

3G

4G

5G

6G

7G

8G

9G

10G

11G

12

Replicate

37

38

39

40

41

42

43

iRT

(Hut

tenh

ain)

Sco

re

YGQTIRPICLPCTEGTTR - 708.3506+++ YGQTIRPICLPCTEGTTR - 711.6867+++ (heavy) YGQTIRPICLPCTEGTTR

Page 18: Targeted Proteomics Environment

Integrating with External Tools GeneSpring & Skyline Automation Tool

(Agilent) MSstats (Purdue) – Grouped study analysis QuaSAR (Broad Institute) – Response curves,

LOD, LOQ MS1 Probe (Buck Institute) – MS1 quant.

statistics TP28 – Alexandria D’Souza

SRM Collider (IMSB) – Interference probability calculator

Page 19: Targeted Proteomics Environment

External Tool MSstats

Page 20: Targeted Proteomics Environment

Replicate Annotations

Page 21: Targeted Proteomics Environment

Custom Reports

Page 22: Targeted Proteomics Environment

Analysis of reports with R – MSstatsDownstream Analysis with Statistical Tools

Page 23: Targeted Proteomics Environment

Skyline Automation with SkylineRunner

Page 24: Targeted Proteomics Environment

MS/MS Spectral Library Sources Global Proteome Machine NIST Peptide Atlas

Build your own from peptide search results Mascot MaxQuant Andromeda Morpheus Myrimatch / IDPicker OMSSA PRIDE XML Protein Pilot Protein Prospector

Proteome Discoverer (MSF) Scaffold – mzIdentML / MGF Spectrum Mill TPP – pepXML / mzXML files –

Peptide Atlas Waters Mse X! Tandem

Page 25: Targeted Proteomics Environment

Integrating Great Ideas (iRT)

Page 26: Targeted Proteomics Environment

Integrating Great Ideas (mProphet)

TP 499 - MacLean

Page 27: Targeted Proteomics Environment

Skyline Team Nick Shulman

Don Marsh

Kaipo Tamura

Danny Broudy Trevor Killeen

Vagish Sharma

Josh Eckels

Greg Taylor

Jarrett Egertson Dario Amodei

Page 28: Targeted Proteomics Environment

Collaborators: U. of Wa.

Eva Baker Jarrett Egertson Jimmy Eng Andrew Stergachis

Biognosys Lukas Reiter Oliver Rinner Claudia Escher

Broad Institute Sue Abbatiello Steve Carr Jake Jaffe D. R. Mani

Buck Institute Birgit Schilling Matthew Rardin Brad Gibson

Duke Will Thompson Arthur Moseley

IMSB Rudolph Aebersold Christina Ludwig Olga Schubert Hannes Röst Lucia Espona Pernas

Purdue Veavi Chang Meena Choi Olga Vitek

Stanford Dario Amodei Parag Mallick

Vanderbilt Matthew Chambers Daniel Liebler David Tabb

Page 29: Targeted Proteomics Environment

Instrument Vendor Support Agilent

Technologies Christine Miller Juli Salcedo Shripad Torvi Yinghang Yang

Bruker Carsten Baessmann Marius Kallhardt Stephanie Kaspar

AB Sciex David Cox Christie Hunter Brent Lefebvre

Thermo-Scientific Markus Kellmann Andreas Kuehn Vlad Zabrouskov

Waters Laurence Firth James Langridge Roy Martin Kieran Neeson Keith Richards