Proteomics analysis: Basics and Applications

Proteomic Analysis:Proteomic Analysis:

Basics and ApplicationsBasics and Applications

Ignasi Forné

February 14, 2013

Introduction

GenomicsGenomics

Introduction

TranscriptomicsTranscriptomics

Introduction

ProteomicsProteomics

Introduction

Genomics Transcriptomics ProteomicsGenomics Transcriptomics Proteomics

Location, Time, Interaction partners, PTMs, Dynamics, Turnover

Adapted from Hein et al, Handbook of System biology 2013

Proteomics: Basics

FractionationEnrichment


Cells/Tissue Proteins Peptides

Proteomics: Basics




C18‐HPLC nESI

Mass Spectrometer (m/z)

Time

Proteomics: Basics




nESIC18‐HPLC

Mass Spectrometer (m/z)

Time

Proteomics: Basics

DDAnESIC18‐HPLCFull MS

MS2 top 1

MS2 top 2

MS2 top 3MS2 p

MS2 ....

MS2 top n-1

MS2 top nMass Spectrometer (m/z)

TimeMS

Time

Full MS

MS2 MS2 MS2 MS2

T: FTMS + p NSI Full ms [300.00-2000.00]

80

90

100472.7700

p [ ]

70

80

90

100

ce

472.7700_ _ _

F: ITMS + c NSI d w Full ms2 [email protected]

90

100575.30

428.25

top 2 top 3 top ntop 1

50

60

70

ve A

bund

ance

944.5325513.2991

769 445330

40

50

60

Rel

ativ

e A

bund

anc

473.2710

50

60

70

80

Abu

ndan

ce20

30

40

Rel

ativ 769.4453

691.3953

1046.2464472 473 474

/

0

10

20 473.7723

474.2738471.8120472.3233

10

20

30

40

Rel

ativ

e

703.36517.17

343.35242.05232.18 713.30686 31

400 600 800 1000 1200 1400 1600 1800 2000m/z

0

10 1381.78261106.55811844.3618

1537.88721306.7396 1890.9115 200 400 600 800m/z

0

0 713.30686.31873.53

Time

Proteomics: Basics


MS2 top 1

MS2 top 2

MS2 top 3MS2 p

MS2 ....

MS2 top n-1


TimeMS

Time

Full MS

MS2 MS2 MS2 MS2


80

90

100472.7700 top 2 top 3 top ntop 1

F: ITMS + c NSI d w Full ms2 [email protected]

90

1001108.53

70

80

90

100

e

944.5324

50

60

70

ve A

bund

ance

944.5325513.2991

769 4453 50

60

70

80

Abu

ndan

ce

810.08

761.14

30

40

50

60

70

Rel

ativ

e A

bund

anc

945.5353

945.0824

944.2667

20

30

40

Rel

ativ 769.4453

691.3953

1046.2464 10

20

30

40

50

Rel

ativ

e A

621.24

1266.51

353.26

915.90493 13 1469 38

944 945 946 947m/z

0

10

20946.5384

9

945.7967

943.5265

400 600 800 1000 1200 1400 1600 1800 2000m/z

0

10 1381.78261106.55811844.3618

1537.88721306.7396 1890.9115

Time

500 1000 1500m/z

0

10 915.90493.13 1469.381714.74

Proteomics: Basics


MS2 top 1

MS2 top 2

MS2 top 3MS2 p

MS2 ....

MS2 top n-1


TimeMS

Time

Full MS

MS2 MS2 MS2 MS2


80

90

100472.7700 top 2 top 3 top ntop 1

60

70

80

90

100

danc

e

513.2990

513.6329F: ITMS + c NSI d w Full ms2 [email protected]

90

100534.53

598.58

50

60

70

ve A

bund

ance

944.5325513.2991

769 445320

30

40

50

60

Rel

ativ

e A

bund

513.9668

50

60

70

80

Abu

ndan

ce

655 13

20

30

40

Rel

ativ 769.4453

691.3953

1046.2464

513.0 513.5 514.0 514.5m/z

0

10514.3010

514.6352512.7917513.1269

10

20

30

40

Rel

ativ

e 655.13

449.42

230.02 717.35343 04

400 600 800 1000 1200 1400 1600 1800 2000m/z

0

10 1381.78261106.55811844.3618

1537.88721306.7396 1890.9115

Time

200 400 600 800 1000m/z

0

10 343.04825.52 926.25 1050.54

Proteomics: Basics


MS2 top 1

MS2 top 2

MS2 top 3MS2 p

MS2 ....

MS2 top n-1


TimeMS

Time

DDA Video

Proteomics: Basics

Peptide charge and mass

p [ ](M+2H)2+ The charge

p g

90

100472.7700 (m2-m1)/z= 0.5 z=2

[m2-m1 = 1]

70

80

ance

z=2 m= M+2H+

0

50

60

ve A

bund

a

473.2710The mass

m/z= (M+2H+)/2

20

30

40

Rel

ativ m/z= (M+2H+)/2

m/z=472.7700

H+ =1 0073

0

10

20 473.7723

474.2738471.8120472.3233

H =1.0073

M= 943 5254472 473 474

/

0

m/z

M 943.5254

Proteomics: Basics

_ _ __ _ _F: ITMS + c NSI d w Full ms2 [email protected]

100575.30

428.25

80

90

60

70

unda

nce

40

50

lativ

e A

bu

20

30Rel 703.36

517.17343.35242.05

0

10 232.18 713.30686.31873.53

200 400 600 800m/z

Proteomics: Basics

y5

danc

e

y4

y5

lative

Abun

d

y2

y3

y4

y

y7b3

b

Rel y2 y6

b2

b4b5

b6

b7

m/z

Proteomics: Basics

Ser Ile Val Lys

Gly Val Met Glu

y5

danc

e

y4

y5

lative

Abun

d

y2

y3

y4

y

y7b3

b

Rel y2 y6

b2

b4b5

b6

b7

m/z

Proteomics: Basics

Ser Ile Val Lys

Gly Val Met Glu

danc

e

Ile

Met Val

GluValGly Ser

IleValGly Ser

Met ValIleValGly Ser

lative

Abun

d

b3

bGly Ser

ValGly SerValGly Ser

MetIleValGly Ser

Rel

b2

b4b5

b6

b7

Gly

m/z

Proteomics: Basics

Ser Ile Val Lys

Gly Val Met Glu

Ile Met Val Glu Lys

Ser

Val

Met

Gl L

Val Glu Lys Val Ile Met Val Glu Lys

danc

e

y4

y5

Val

Glu

Val

Lys

Glu Lys Ile Met Val Glu Lys

lative

Abun

d

y2

y3

y4

y

y7

Rel y2 y6

m/z

Proteomics: Basics

Ser Ile Val Lys

Gly Val Met Glu

y5

danc

e

y4

y5

Ile

lative

Abun

d

y2

y3

y4

y

y7b3

b

SerVal

Met

Val

Rel y2 y6

b2

b4b5

b6

b7

MetIle

ValGluSer

m/z

Proteomics: Basics


MS2 top 1

MS2 top 2

MS2 top 3MS2 p

MS2 ....

MS2 top n-1


TimeMS

Time

The ChargeThe Mass of the Peptide

The Masses of the Fragments

Proteomics: Basics


MS2 top 1

MS2 top 2

MS2 top 3MS2 p

MS2 ....

MS2 top n-1


TimeMS

Time

Peptide identification Protein identification

Rel

ativ

e Ab

unda

nce Sequence

Database Search

m/z

R Search

Score Calculation

Target-Decoy-based

FDR th h ldiPeptide to protein mapping

Adapted from Hein et al, Handbook of System biology 2013

FDR thresholdingFDR thresholding

Proteomics: Basics

Peptide QuantificationnESIC18‐HPLCPeptide Quantification

p [ ]

70

80

90

100

ance

472.7700

Intensity Mass Spectrometer (m/z)0

10

20

30

40

50

60

Rel

ativ

e A

bund

a

473.2710

473.7723

474.2738471.8120472.3233

Peptide 1 L Sample A

Intensity Mass Spectrometer (m/z)472 473 474

/

0

Time

m/z

Proteomics: Basics

Peptide QuantificationPeptide Quantification

IntensityIntensity


Time

m/z

Proteomics: Basics

Peptide Quantification

Label Free(across runs)

Peptide Quantification

Intensity

( )

Peptide 1 L Sample B

Intensity


Time

Peptide 1 H Sample Brun 2run 2

Isotopic Labelling(SILAC Dimethyl

run 1MS2 Reporter Ions

m/z

(SILAC, Dimethyl, Heavy peptides)

Proteomics: Basics

Ong et al, Nat Chem Biol 2005

Proteomics: Basics

de Godoy et al, Nature 2008

Proteomics: Basics

In depth analysisIn depth analysis

Michalski et al, J Proteome Res 2011Cox et al, Ann Rev Biochemistry 2011

Proteomics: Applications

Expression Proteomics

WT WT+KO Protein Identification

Lys

In‐gel digestion

KO

LC‐MSMS

+

Quantification SILAC

PINK1 KO

Lys +8

SILAC

2

1

1.5

se

Keratin

BSA

WT > KO

0

0.5

-2 -1.5 -1 -0.5 0 0.5 1 1.5

g 10

Rat

io H

/L R

ever

s

Serie1

-1

-0.5

log

-1.5

log 10 Ratio H/L Forward

KO> WT

Schreiner et al, Mol Biol Cell 2012


Interactomics

Protein Identification

Arg, Lys

Protein Extract 1

‐/+P

+

Quantification SILAC

Arg +10, Lys +8

Protein Extract 2

P

In‐gel digestion LC‐MSMS

SILACdigestion

2

Proteins2

ProteinsINTERACTORS

0 5

1

1.5

erse

Proteins confirmed in both SILAC

1 and 6

(SIL

AC 6

)

0 5

1

1.5

erse

Proteins confirmed in both SILAC

1 and 6

(SIL

AC 6

)

INTERACTORS

-0.5

0

0.5

-1.5 -1.25 -1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 1.25 1.5

og 1

0 R

atio

H/L

Rev

e

Serie1

-0.5

0

0.5

-1.5 -1.25 -1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 1.25 1.5

og 1

0 R

atio

H/L

Rev

e

Serie1

-1.5

-1

lo

-1.5

-1

lo

-2

log 10 Ratio H/L Forward (SILAC 1)-2

log 10 Ratio H/L Forward (SILAC 1)


PTM Analysis

GG‐K Identification

GGUb Ligase

+

QuantificationIn‐gel digestion LC‐MSMS

GG

GG

DCC complexGG

Villa et al, Mol Cell 2012


PTM Analysis

ControlP

Protein Extract

P‐site Identification

+ LC‐MSMSPhosphopeptide enrichment

PIn‐solutiondigestion

P

l

Quantification Labelfree

Protein Extract P

P

P

P

Stimulus P

Ac‐K

Identification AcProtein Extract

AcAc

+

Quantification

AcExtract

Protein Extract

In‐solutiondigestion

Ac

LC‐MSMSAcetylpeptide enrichment

LabelfreeExtract

Ac

Ac

Proteomics: Missing values

Proteome

run 1 run 2 run 3 run 4

1

Time Course

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

5

Protein

10

...

Proteomics

Nature Methods: Method of the Year 2012

Proteomics: Targeted Analysis

Proteome

run 1 run 2 run 3 run 4

Ti C

1

Time Course

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

5

Protein

10

...


Ser Ile Val Lys

Gly Val Met Glu

y5

danc

e

y4

y5

lative

Abun

d

y2

y3

y4

y

y7b3

b

Rel y2 y6

b2

b4b5

b6

b7

m/z


Ser Ile Val Lys

Gly Val Met Glu

Ile Met Val Glu Lys

danc

e

Val

Met

Gl L

Val Glu Lys

y4

y5

lative

Abun

d Val Glu Lys

y3

y4

Rel

m/z


p [ ]472 7700

ce

Met

Ile

Val Glu Lys

Met Val Glu Lys

y580

90

100472.7700

ve Ab

unda

nc

Val Glu Lys

y3

y4

40

50

60

70

ve A

bund

ance

473.2710

Relat

iv

10

20

30

40

Rel

ativ

473.7723

472 3233

472 473 474/

0

10474.2738471.8120

472.3233

m/z

Picotti et al, Nat Methods 2012SRM Video


Gallien et al, J Mass Spectrometry 2011


Picotti et al, Cell 2009

Structural Proteomics

Hein et al, Handbook of System biology 2013

Structural Proteomics

Examples

Herzog et al Science 2012

Summary

• MS‐based Proteomics:

• Identification

Q ifi i• Quantification

• Shotgun/DiscoveryShotgun/Discovery

• Targeted

• Structural Proteomics and Interactomics

Thanks!!

Prof. Axel ImhofAnd the Colleagues at the Imhof‘s and the ZfP

(Molecular Biology Dept. Biomedical Center‐LMU)

Questions??

Proteomic Analysis:Proteomic Analysis:

Basics and ApplicationsBasics and Applications

Ignasi Forné

February 14, 2013

Proteomics analysis: Basics and Applications

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