spatial proteomics combining experimental and annotation ...€¦ · spatial proteomics combining...
TRANSCRIPT
Spatial proteomicsCombining experimental and annotation data to
predict protein sub-cellular localisation.
Laurent [email protected] – @lgatt0
Computational Proteomics Unithttp://cpu.sysbiol.cam.ac.uk/
University of Cambridge
13 Jan 2015, Heidelberg
Plan
Introduction
Spatial proteomics
Previously . . .
Transfer learning
Regulations
Cell organisation
Spatial proteomics is the systematic study of protein localisations.
Image from Wikipedia http://en.wikipedia.org/wiki/Cell_(biology).
Spatial proteomics - Why?
Mis-localisationDisruption of the targeting/trafficking process alters propersub-cellular localisation, which in turn perturb the cellular functionsof the proteins.I Abnormal protein localisation leading to the loss of functional
n effects in diseases (Laurila and Vihinen, 2009).I Disruption of the nuclear/cytoplasmic transport (nuclear
pores) have been detected in many types of carcinoma cells(Kau et al., 2004).
Multi- and re-localisation
I Differentiation: Tfe3 in mouse ESC (Betschinger et al., 2013).I Metabolism: changes in carbon sources, elemental limitations.
Plan
Introduction
Spatial proteomics
Previously . . .
Transfer learning
Spatial proteomics - How, experimentally
Single celldirect
observation
Population level
Subcellular fractionation (number of fractions)
Tagging Quantitative mass spectrometryCataloguing Relative abundance
1 fraction2 fractions(enriched
and crude)n discrete fractions
n continuous fractions(gradient approaches)
Subtractiveproteomics
(enrichment)
Invariantrich
fraction(clustering)
(χ )2PCP LOPIT
(PCA, PLS-DA)
Pure fraction
catalogue
GFPEpitope
Prot.-spec.antibody
Figure : Organelle proteomics approaches (Gatto et al., 2010)
Fusion proteins and immunofluorescence
Spatial proteomics - How, experimentally
Single celldirect
observation
Population level
Subcellular fractionation (number of fractions)
Tagging Quantitative mass spectrometryCataloguing Relative abundance
1 fraction2 fractions(enriched
and crude)n discrete fractions
n continuous fractions(gradient approaches)
Subtractiveproteomics
(enrichment)
Invariantrich
fraction(clustering)
(χ )2PCP LOPIT
(PCA, PLS-DA)
Pure fraction
catalogue
GFPEpitope
Prot.-spec.antibody
Figure : Organelle proteomics approaches (Gatto et al., 2010). Gradientapproaches: Dunkley et al. (2006), Foster et al. (2006).
⇒ Explorative/discovery approches, global localisation maps.
Fractionation/centrifugation
Quantitation/identificationby mass spectrometry
e.g. Mitochondrion
Cell lysis
e.g. Mitochondrion
Quantitation data and organelle markers
Fraction1 Fraction2 . . . Fractionm markersp1 q1,1 q1,2 . . . q1, m unknownp2 q2,1 q2,2 . . . q2, m loc1
p3 q3,1 q3,2 . . . q3, m unknownp4 q4,1 q4,2 . . . q4, m loci...
......
......
...
pj qj,1 qj,2 . . . qj, m unknown
Visualisation and classification
0.2
0.3
0.4
0.5
Correlation profile − ER
Fractions
1 2 4 5 7 81112
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Correlation profile − Golgi
Fractions
1 2 4 5 7 81112
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Correlation profile − mit/plastid
Fractions
1 2 4 5 7 81112
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0.35
Correlation profile − PM
Fractions
1 2 4 5 7 81112
0.1
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0.5
0.6
Correlation profile − Vacuole
Fractions
1 2 4 5 7 81112
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−10 −5 0 5
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05
Principal component analysis
PC1
PC
2
●
ERGolgimit/plastidPM
vacuolemarkerPLS−DAunknown
Figure : From Gatto et al. (2010), Arabidopsis thaliana data from Dunkleyet al. (2006)
Plan
Introduction
Spatial proteomics
Previously . . .
Transfer learning
Previously . . .
I Infrastructure: MSnbase(Gatto and Lilley, 2012)
I Spatial proteomics data:pRolocdata (Gatto et al.,2014)
I Novely detection:pRoloc::phenoDisco
(Breckels et al., 2013)I Localisation prediction and
visualisation: pRoloc (Gattoet al., 2014) and pRolocGUI
MSnSet (storageMode: lockedEnvironment)
assayData: 2031 features, 8 samples
element names: exprs
protocolData: none
phenoData
sampleNames: n113 n114 ... n121 (8 total)
varLabels: Fraction.information
varMetadata: labelDescription
featureData
featureNames: Q62261 Q9JHU4 ... Q9EQ93 (2031 total)
fvarLabels: Uniprot.ID UniprotName ... markers (8 total)
fvarMetadata: labelDescription
experimentData: use 'experimentData(object)'Annotation:
- - - Processing information - - -
Loaded on Fri Nov 7 16:49:05 2014.
Normalised to sum of intensities.
Added markers from 'mrk' marker vector. Fri Nov 7 16:49:05 2014
MSnbase version: 1.13.16
Previously . . .
I Infrastructure: MSnbase(Gatto and Lilley, 2012)
I Spatial proteomics data:pRolocdata (Gatto et al.,2014)
I Novely detection:pRoloc::phenoDisco
(Breckels et al., 2013)I Localisation prediction and
visualisation: pRoloc (Gattoet al., 2014) and pRolocGUI
I Several mouse E14TG2a Embryonic Stem cells.I Human Embryonic Kidney fibroblast cells.I The Arabidopsis AT CHLORO data base (Ferro et
al., 2010).I Mouse organs (Foster et al., 2006).I Arabidopsis from callus (Dunkley et al., 2006;
Nikolovksi et al. 2014) and roots (Groen et al.,2014).
I Drosophila embryos (Tan et al., 2009).I Chicken DT40 Lymphocyte cell (Hall et al., 2009).I . . .I Collected from the literature
Previously . . .
I Infrastructure: MSnbase(Gatto and Lilley, 2012)
I Spatial proteomics data:pRolocdata (Gatto et al.,2014)
I Novely detection:pRoloc::phenoDisco
(Breckels et al., 2013)I Localisation prediction and
visualisation: pRoloc (Gattoet al., 2014) and pRolocGUI
−3 −2 −1 0 1 2 3
−3
−2
−1
01
23
PC1 (58.53%)
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ERGolgimitochondrionPMunknown
−3 −2 −1 0 1 2 3
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CytoskeletonERGolgiLysosomemitochondrionNucleus
PeroxisomePMProteasomeRibosome 40SRibosome 60Sunknown
Previously . . .
I Infrastructure: MSnbase(Gatto and Lilley, 2012)
I Spatial proteomics data:pRolocdata (Gatto et al.,2014)
I Novely detection:pRoloc::phenoDisco
(Breckels et al., 2013)I Localisation prediction
and visualisation: pRoloc(Gatto et al., 2014) andpRolocGUI
−3 −2 −1 0 1 2 3
−3
−2
−1
01
23
PC1 (58.53%)P
C2
(29.
96%
)
CytoskeletonERGolgiLysosomemitochondrionNucleus
PeroxisomePMProteasomeRibosome 40SRibosome 60S
Plan
Introduction
Spatial proteomics
Previously . . .
Transfer learning
What about annotation data from repositories such as GO,sequence features, signal peptide, transmembrane domains,images, protein-protein interactions, ... . . .
I From a user perspective: ”free/cheap” vs. expensiveI Abundant (all proteins, 100s of features) vs. (experimentally)
limited/targeted (1000s of proteins, 6 – 20 of features)I For localisation in system at hand: low vs. high qualityI Static vs. dynamic
number GO features� experimental fractions⇒ dilution of experimental data
What about annotation data from repositories such as GO,sequence features, signal peptide, transmembrane domains,images, protein-protein interactions, ... . . .
I From a user perspective: ”free/cheap” vs. expensiveI Abundant (all proteins, 100s of features) vs. (experimentally)
limited/targeted (1000s of proteins, 6 – 20 of features)I For localisation in system at hand: low vs. high qualityI Static vs. dynamic
number GO features� experimental fractions⇒ dilution of experimental data
GoalSupport/complement the primary target domain (experimentaldata) with auxiliary data (annotation) features withoutcompromising the integrity of our primary data.
Updated experimental design for
I primary/experimental data
and
I auxiliary/annotation data
Fractionation/centrifugation
Quantitation/identificationby mass spectrometry
Database query
Extract GO CC terms
Convert terms to binary
PR
IMA
RY EX
PER
IMEN
TAL
DATA
AU
XIL
IARY D
RY D
ATA
O00767P51648Q2TAA5Q9UKV5......
GO:0016021 GO:0005789 GO:0005783 ... ... ...
1 1 1 ... ... ...1 1 0 ... ... ...1 1 0 ... ... ...0 0 0 ... ... .... . .. . .. . .. . .. . .. . .
x1
.
.
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.
.
.xn
GO1 ... ... ... ... GOA
O00767P51648Q2TAA5Q9UKV5......
0.1361 0.150 0.1062 0.147 0.277 0.1429 0.0380 0.003380.1914 0.205 0.0566 0.165 0.237 0.0996 0.0180 0.027270.1297 0.201 0.0546 0.146 0.292 0.1463 0.0206 0.009020.0939 0.207 0.0419 0.204 0.344 0.1098 0.0000 0.00000. . . . . . . .. . . . . . . .. . . . . . . .. . . . . . . .. . . . . . . .. . . . . . . .
x1
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.
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.
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.xn
X113 X114 X115 X116 X117 X118 X119 X121
Visualisation Visualisation
e.g. Mitochondrion
Cell lysis
e.g. Mitochondrion
−2 0 2 4
−2
−1
01
23
4
PC1 (40.28%)
PC
2 (2
5.7%
)
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40S Ribosome60S RibosomeCytosolEndoplasmic reticulumLysosomeMitochondrionNucleus − ChromatinNucleus − NucleolusPlasma membraneProteasomeunknown
Data from mouse stem cells (E14TG2a)
We use a class-weighted kNNtransfer learning algorithm tocombine primary and auxiliarydata, based on Wu andDietterich (2004):
V(ci)j = θ∗nPij + (1 − θ∗)nA
ij
Classes and weightsC = {ci=1 , . . . , ci=l }; Θ = {0, 0.5, 1}
Primary data
LP =
q1,1 q1,2 . . . q1,mq2,1 q2,2 . . . q2,m
.
.
.
.
.
.qj,1 qj,2 . . . qj,m
;y1y2
.
.
.yj
; kP
Auxiliary data
LA =
b1,1 b1,2 . . . . . . b1,nb2,1 b2,2 . . . . . . b2,n
.
.
.
.
.
.bj,1 bj,2 . . . . . . bj,n
;y1y2
.
.
.yj
; kA
Neighbour matrices
NP =
ci=1 . . . ci=l
nP1,1 . . . nP
1,lnP
2,1 . . . nP2,l
.
.
.
.
.
.
; NA =
ci=1 . . . ci=l
nA1,1 . . . nA
1,lnA
2,1 . . . nA2,l
.
.
.
.
.
.
Classes and weightsC = {ci=1 , . . . , ci=l }; Θ = {0, 0.5, 1}
Primary data
LP =
q1,1 q1,2 . . . q1,mq2,1 q2,2 . . . q2,m
.
.
.
.
.
.qj,1 qj,2 . . . qj,m
;y1y2
.
.
.yj
; kP
Auxiliary data
LA =
b1,1 b1,2 . . . . . . b1,nb2,1 b2,2 . . . . . . b2,n
.
.
.
.
.
.bj,1 bj,2 . . . . . . bj,n
;y1y2
.
.
.yj
; kA
Neighbour matrices
NP =
ci=1 . . . ci=l
nP1,1 . . . nP
1,lnP
2,1 . . . nP2,l
.
.
.
.
.
.
; NA =
ci=1 . . . ci=l
nA1,1 . . . nA
1,lnA
2,1 . . . nA2,l
.
.
.
.
.
.
1
2
●
●
●
c1c2c3
NP =
c1 c2 c3
p133 0 0
p213
23 0
......
...
Classes and weightsC = {ci=1 , . . . , ci=l }; Θ = {0, 0.5, 1}
Primary data
LP =
q1,1 q1,2 . . . q1,mq2,1 q2,2 . . . q2,m
.
.
.
.
.
.qj,1 qj,2 . . . qj,m
;y1y2
.
.
.yj
; kP
Auxiliary data
LA =
b1,1 b1,2 . . . . . . b1,nb2,1 b2,2 . . . . . . b2,n
.
.
.
.
.
.bj,1 bj,2 . . . . . . bj,n
;y1y2
.
.
.yj
; kA
Neighbour matrices
NP =
ci=1 . . . ci=l
nP1,1 . . . nP
1,lnP
2,1 . . . nP2,l
.
.
.
.
.
.
; NA =
ci=1 . . . ci=l
nA1,1 . . . nA
1,lnA
2,1 . . . nA2,l
.
.
.
.
.
.
Weights matrix (labelled)
c1 c2 c3
θ1 0 0 0θ2 0 0 1
θi...
...... 1 1 0θΘl 1 1 1
F11
F12
F1i...
F1Θl
θ∗ = {1, 0, 1}
(r BiocParallel)
Classes and weightsC = {ci=1 , . . . , ci=l }; Θ = {0, 0.5, 1}
Primary data
LP =
q1,1 q1,2 . . . q1,mq2,1 q2,2 . . . q2,m
.
.
.
.
.
.qj,1 qj,2 . . . qj,m
;y1y2
.
.
.yj
; kP
Auxiliary data
LA =
b1,1 b1,2 . . . . . . b1,nb2,1 b2,2 . . . . . . b2,n
.
.
.
.
.
.bj,1 bj,2 . . . . . . bj,n
;y1y2
.
.
.yj
; kA
Neighbour matrices
NP =
ci=1 . . . ci=l
nP1,1 . . . nP
1,lnP
2,1 . . . nP2,l
.
.
.
.
.
.
; NA =
ci=1 . . . ci=l
nA1,1 . . . nA
1,lnA
2,1 . . . nA2,l
.
.
.
.
.
.
Class-weighted classifier(unlabelled)
V(ci)j = θ∗nPij + (1 − θ∗)nA
ij
ci=1 . . . ci=l
123 V(ci)j...
j
yj = argmax(V(ci)j)
θ∗ = {1, 0, 1} NP =
c1 c2 c3
p133 0 0
p213
23 0
......
...
V(c1)1 =1 ×
33
+ (1 − 1) × nA1,1
V(c2)1 =0 × 0 + (1 − 0) × nA1,2
V(c3)1 =1 × 0 + (1 − 1) × nA1,3
V(c1)2 =1 ×13
+ (1 − 1) × nA1,1
V(c2)2 =0 ×23
+ (1 − 0) × nA1,2
V(c3)2 =1 × 0 + (1 − 1) × nA1,3
Class-weighted classifier(unlabelled)
V(ci)j = θ∗nPij + (1 − θ∗)nA
ij
c1 c2 c3
1 V(c1)1 V(c2)1 V(c3)1
2 V(c1)2 V(c2)2 V(c3)2...
...
j
yj = argmax(V(ci)j)
D E
A B C
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●
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●●
40S Ribosome 60S Ribosome Cytosol Endoplasmic reticulum
Lysosome Mitochondrion Nucleus − Chromatin Nucleus − Nucleolus
Plasma membrane Proteasome
0.4
0.6
0.8
1.0
0.6
0.7
0.8
0.9
1.0
0.00
0.25
0.50
0.75
1.00
0.7
0.8
0.9
1.0
0.00
0.25
0.50
0.75
1.00
0.75
0.80
0.85
0.90
0.95
1.00
0.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
0.25
0.50
0.75
1.00
0.00
0.25
0.50
0.75
1.00
Combined Primary Auxiliary Combined Primary Auxiliary Combined Primary Auxiliary Combined Primary Auxiliary
Combined Primary Auxiliary Combined Primary Auxiliary Combined Primary Auxiliary Combined Primary Auxiliary
Combined Primary Auxiliary Combined Primary Auxiliary
F1 s
core
−6 −4 −2 0
−6−4
−20
2
PC1 (3.43%)
PC2
(2.0
8%)
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40S Ribosome60S RibosomeCytosolEndoplasmic reticulumLysosomeMitochondrionNucleus − ChromatinNucleus − NucleolusPlasma membraneProteasomeunknown
−2 0 2 4
−2−1
01
23
4
PC1 (40.28%)
PC2
(25.
7%)
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40S Ribosome60S RibosomeCytosolEndoplasmic reticulumLysosomeMitochondrionNucleus − ChromatinNucleus − NucleolusPlasma membraneProteasomeunknown ●
●
0.5
0.6
0.7
0.8
0.9
Combined Primary Auxiliary
F1 s
core
Proteasome
Plasma membrane
Nucleus − Nucleolus
Nucleus − Chromatin
Mitochondrion
Lysosome
Endoplasmic reticulum
Cytosol
60S Ribosome
40S Ribosome
0 1/3 2/3 1Classifier weight
Cla
ss
Data from mouse stem cells (E14TG2a).
Why? – Dual-localisation Proteins may be presentsimultaneously in several organelles (e.g. trafficking).
−6 −4 −2 0 2 4 6
−4
−2
02
4
PC1 (64.36%)
PC
2 (2
2.34
%) ●
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ER lumenER membraneGolgiMitochondrionPlastidPMRibosomeTGNvacuoleunknown
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From Betschinger et al. (2013)
−6 −4 −2 0 2 4
−4
−2
02
4
Mouse ESC (E14TG2a) in serum LIF
PC1 (50.05%)
PC
2 (2
4.61
%)
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Actin cytoskeletonCytosolEndosomeER/GAExtracellular matrixLysosomeMitochondriaNucleus − ChromatinNucleus − NucleolusPeroxisomePlasma MembraneProteasomeRibosome 40SRibosome 60Sunknown
●Tfe3
Why? – Dual-localisation Proteins may be presentsimultaneously in several organelles (e.g. trafficking).
−6 −4 −2 0 2 4 6
−4
−2
02
4
PC1 (64.36%)
PC
2 (2
2.34
%) ●
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ER lumenER membraneGolgiMitochondrionPlastidPMRibosomeTGNvacuoleunknown
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From Betschinger et al. (2013)
−6 −4 −2 0 2 4
−4
−2
02
4
Mouse ESC (E14TG2a) in serum LIF
PC1 (50.05%)
PC
2 (2
4.61
%)
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Actin cytoskeletonCytosolEndosomeER/GAExtracellular matrixLysosomeMitochondriaNucleus − ChromatinNucleus − NucleolusPeroxisomePlasma MembraneProteasomeRibosome 40SRibosome 60Sunknown
●Tfe3
FundingBBSRC and PRIME-XS EU FP7
I Lisa Breckels, Computational Proteomics UnitI Kathryn Lilley, Cambridge Centre of ProteomicsI Sean Holden, Computer Laboratory
Thank you for your attention
J Betschinger, J Nichols, S Dietmann, P D Corrin, P J Paddison, and A Smith. Exit from pluripotency is gated byintracellular redistribution of the bhlh transcription factor tfe3. Cell, 153(2):335–47, Apr 2013. doi:10.1016/j.cell.2013.03.012.
LM Breckels, L Gatto, A Christoforou, AJ Groen, KS Lilley, and MW Trotter. The effect of organelle discovery uponsub-cellular protein localisation. J Proteomics, 88:129–40, Aug 2013.
TPJ Dunkley, S Hester, IP Shadforth, J Runions, T Weimar, SL Hanton, JL Griffin, C Bessant, F Brandizzi, C Hawes,RB Watson, P Dupree, and KS Lilley. Mapping the Arabidopsis organelle proteome. PNAS, 103(17):6518–6523, Apr2006.
LJ Foster, CL de Hoog, Y Zhang, Y Zhang, X Xie, VK Mootha, and M Mann. A mammalian organelle map by proteincorrelation profiling. Cell, 125(1):187–199, Apr 2006.
L Gatto and KS Lilley. MSnbase - an R/Bioconductor package for isobaric tagged mass spectrometry data visualization,processing and quantitation. Bioinformatics, 28(2):288–9, Jan 2012.
L Gatto, JA Vizcaino, H Hermjakob, W Huber, and KS Lilley. Organelle proteomics experimental designs and analysis.Proteomics, 2010.
L Gatto, L M Breckels, S Wieczorek, T Burger, and K S Lilley. Mass-spectrometry based spatial proteomics data analysisusing pRoloc and pRolocdata. Bioinformatics, Jan 2014.
TR Kau, JC Way, and PA Silver. Nuclear transport and cancer: from mechanism to intervention. Nat Rev Cancer, 4(2):106–17, Feb 2004.
K Laurila and M Vihinen. Prediction of disease-related mutations affecting protein localization. BMC Genomics, 10:122,2009.
P Wu and TG Dietterich. Improving svm accuracy by training on auxiliary data sources. In Proceedings of the Twenty-firstInternational Conference on Machine Learning, ICML ’04, New York, NY, USA, 2004. ACM.