proteomics analysis: basics and applications
TRANSCRIPT
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Proteomic Analysis:Proteomic Analysis:
Basics and ApplicationsBasics and Applications
Ignasi Forné
February 14, 2013
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Introduction
GenomicsGenomics
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Introduction
TranscriptomicsTranscriptomics
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Introduction
ProteomicsProteomics
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Introduction
Genomics Transcriptomics ProteomicsGenomics Transcriptomics Proteomics
Location, Time, Interaction partners, PTMs, Dynamics, Turnover
Adapted from Hein et al, Handbook of System biology 2013
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Proteomics: Basics
FractionationEnrichment
FractionationEnrichment
Cells/Tissue Proteins Peptides
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Proteomics: Basics
FractionationEnrichment
FractionationEnrichment
Cells/Tissue Proteins Peptides
C18‐HPLC nESI
Mass Spectrometer (m/z)
Time
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Proteomics: Basics
FractionationEnrichment
FractionationEnrichment
Cells/Tissue Proteins Peptides
C18‐HPLC nESI
Mass Spectrometer (m/z)
Time
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Proteomics: Basics
FractionationEnrichment
FractionationEnrichment
Cells/Tissue Proteins Peptides
nESIC18‐HPLC
Mass Spectrometer (m/z)
Time
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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
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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 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
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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 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
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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
DDA Video
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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
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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
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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
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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
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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
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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
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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
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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
The ChargeThe Mass of the Peptide
The Masses of the Fragments
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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
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
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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
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Proteomics: Basics
Peptide QuantificationPeptide Quantification
IntensityIntensity
Peptide 1 L Sample A
Time
m/z
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Proteomics: Basics
Peptide Quantification
Label Free(across runs)
Peptide Quantification
Intensity
( )
Peptide 1 L Sample B
Intensity
Peptide 1 L Sample A
Time
Peptide 1 H Sample Brun 2run 2
Isotopic Labelling(SILAC Dimethyl
run 1MS2 Reporter Ions
m/z
(SILAC, Dimethyl, Heavy peptides)
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Proteomics: Basics
Ong et al, Nat Chem Biol 2005
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Proteomics: Basics
de Godoy et al, Nature 2008
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Proteomics: Basics
In depth analysisIn depth analysis
Michalski et al, J Proteome Res 2011Cox et al, Ann Rev Biochemistry 2011
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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
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Proteomics: Applications
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)
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Proteomics: Applications
PTM Analysis
GG‐K Identification
GGUb Ligase
+
QuantificationIn‐gel digestion LC‐MSMS
GG
GG
DCC complexGG
Villa et al, Mol Cell 2012
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Proteomics: Applications
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
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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
...
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Proteomics
Nature Methods: Method of the Year 2012
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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
...
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Proteomics: Targeted Analysis
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
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Proteomics: Targeted Analysis
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
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Proteomics: Targeted Analysis
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
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Proteomics: Targeted Analysis
Gallien et al, J Mass Spectrometry 2011
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Proteomics: Targeted Analysis
Picotti et al, Cell 2009
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Proteomics: Targeted Analysis
Picotti et al, Cell 2009
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Structural Proteomics
Hein et al, Handbook of System biology 2013
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Structural Proteomics
Examples
Herzog et al Science 2012
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Summary
• MS‐based Proteomics:
• Identification
Q ifi i• Quantification
• Shotgun/DiscoveryShotgun/Discovery
• Targeted
• Structural Proteomics and Interactomics
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Thanks!!
Prof. Axel ImhofAnd the Colleagues at the Imhof‘s and the ZfP
(Molecular Biology Dept. Biomedical Center‐LMU)
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Questions??
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Proteomic Analysis:Proteomic Analysis:
Basics and ApplicationsBasics and Applications
Ignasi Forné
February 14, 2013
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