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Introduction to MS-based proteomics andBioconductor infrastructure
Laurent Gatto
[email protected] � @lgatt0
http://cpu.sysbiol.cam.ac.uk
CSAMA � 17 June 2015
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Outline
Proteomics and MS data
Bioconductor infrastructure
Examples
Ranges infrastructure
Application: spatial proteomics
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Mass-spectrometry � LC-MS/MS
Precursor iondissociation
Detector
Precursor ions MS1
Fragmented ions
MSMS - MS2
AnalyserSourceSeparation
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0 500 1000 1500 2000 2500 3000 3500
020
4060
8010
0Chromatogram: total intensity over time
Time (sec)
Tota
l ion
cur
rent
TMT_Erwinia_1uLSike_Top10HCD_isol2_45stepped_60min_01−20141210.mzMLTIC: 7.22e+09
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MS1 (and MS2) spectra
400 500 600 700 800
0.0e
+00
5.0e
+07
1.0e
+08
1.5e
+08
2.0e
+08
MS1 scan @ 21:3 min
m/z
inte
nsity 200 400 600 800
0e+
002e
+06
4e+
066e
+06
8e+
061e
+07
MS2 scan, precursor m/z 460.79
200 400 600 800 1000
0e+
001e
+06
2e+
063e
+06
4e+
065e
+06
6e+
06
MS2 scan, precursor m/z 488.8
m/z
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Mass-spectrometry � LC-MS/MS
Precursor iondissociation
Detector
Precursor ions MS1
Fragmented ions
MSMS - MS2
AnalyserSourceSeparation
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cid <- calculateFragments("AEGKLRFK",
type=c("b", "y"), z=2)
## Modifications used: C=160.030649
ht(cid, n = 3)
## mz ion type pos z seq
## 1 36.52583 b1 b 1 2 A
## 2 101.04713 b2 b 2 2 AE
## 3 129.55786 b3 b 3 2 AEG
## ...
## mz ion type pos z seq
## 31 357.7185 y6* y* 6 2 GKLRFK
## 32 422.2398 y7* y* 7 2 EGKLRFK
## 33 457.7583 y8* y* 8 2 AEGKLRFK
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MS1 and MS2 spectra
400 500 600 700 800
0.0e
+00
5.0e
+07
1.0e
+08
1.5e
+08
2.0e
+08
MS1 scan @ 21:3 min
m/z
inte
nsity 200 400 600 800
0e+
002e
+06
4e+
066e
+06
8e+
061e
+07
MS2 scan, precursor m/z 460.79
200 400 600 800 1000
0e+
001e
+06
2e+
063e
+06
4e+
065e
+06
6e+
06
MS2 scan, precursor m/z 488.8
m/z
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MS1 and MS2 spectra
400
600
800
1000
1200
21.10
21.15
21.20
21.25
21.30
21.35
0.0e+00
5.0e+07
1.0e+08
1.5e+08
2.0e+08
m/zretention time200
400
600
800
1000
1200
21.06
21.07
21.08
21.09
21.10
21.11
0.0e+00
5.0e+07
1.0e+08
1.5e+08
2.0e+08
m/zretention time
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Proteomics data
I raw data:MS1 and MS2 overretention time
I identi�cation:MS2
I quantitation:MS1 or MS2
I protein database(to match MS2 spectraagainst)
Status package
Raw (mz*ML) X mzR
mzTab X MSnbase
mgf X MSnbase
mzIdentML X mzID, mzRmzQuantML (?mzR)
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Bioconductor infrastructure
biocViews: Proteomics, MassSpectrometry
010
2030
4050
Num
ber
of B
ioco
nduc
tor
pack
ages
2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 2.14
Bioconductor Versions
ProteomicsMassSpectrometryMassSpectrometryData
010
000
2000
030
000
4000
050
000
Pac
kage
dow
nloa
ds
2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 2.14
Bioconductor Versions
ProteomicsMassSpectrometry
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Learning from Bioconductor
| genomics | proteomics |
|------------------+------------------------|
| eSet (past?) | *MSnSet (present) |
| Ranges (present) | *Pbase et al. (future) |
| | PPI |
| | *localisation (present)|
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MSnSet
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Example
library("MSnbase")
rx <- readMSData("rawdata.mzML") ## raw data
rx <- addIdentificationData(rx, "identification.mzid")
rx <- rx[!is.na(fData(rx)$pepseq)]
plot(rx[[10]], reporters = TMT6, full=TRUE)
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Example
0e+00
2e+05
4e+05
6e+05
8e+05
300 600 900 1200M/Z
Inte
nsity
Precursor M/Z 600.36
0e+00
2e+05
4e+05
6e+05
8e+05
126.080 126.591 127.102 127.613 128.124 128.635 129.146 129.657 130.168 130.679 131.190
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Example
library("MSnbase")
rx <- readMSData(f, centroided = TRUE)
rx <- addIdentificationData(rx, g)
rx <- rx[!is.na(fData(rx)$pepseq)]
plot(rx[[10]], reporters = TMT6, full=TRUE)
plot(rx[[4730]], rx[[4929]])
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Example
0 500 1000 1500
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0−
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nsity
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prec scan: 6975, prec mass: 545.017, prec z: 3, # common: 34
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prec scan: 7198, prec mass: 545.017, prec z: 3, # common: 35
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Example
library("MSnbase")
rx <- readMSData(f, centroided = TRUE)
rx <- addIdentificationData(rx, g)
rx <- rx[!is.na(fData(rx)$pepseq)]
plot(rx[[10]], reporters = TMT6, full=TRUE)
plot(rx[[4730]], rx[[4929]])
qt <- quantify(rx, reporters = TMT6)
## qt <- readMSnSet("quantdata.csv", ecols = 5:11)
nqt <- normalise(qt, method = "vsn")
boxplot(exprs(nqt))
MAplot(nqt[, 1:2])
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Example
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3.0 3.5 4.0 4.5−
0.15
−0.
10−
0.05
0.00
0.05
0.10
0.15
A
M
Median: 0.000187IQR: 0.013
![Page 21: Introduction to MS-based proteomics and Bioconductor infrastructure · 2015-06-23 · CSAMA 17 June 2015. Outline Proteomics and MS data Bioconductor infrastructure ... 5.0e+07 1.0e+08](https://reader034.vdocuments.net/reader034/viewer/2022042323/5f0dbd5d7e708231d43bd8f0/html5/thumbnails/21.jpg)
More
I RforProteomics package
library("RforProteomics")
RforProteomics()
RProtVis()
citation(package = "RforProteomics")
I Proteomics work�ow on the Bioc site
I Lab on Friday
![Page 22: Introduction to MS-based proteomics and Bioconductor infrastructure · 2015-06-23 · CSAMA 17 June 2015. Outline Proteomics and MS data Bioconductor infrastructure ... 5.0e+07 1.0e+08](https://reader034.vdocuments.net/reader034/viewer/2022042323/5f0dbd5d7e708231d43bd8f0/html5/thumbnails/22.jpg)
I protein database
I raw dataI quantitationI identi�cation
![Page 23: Introduction to MS-based proteomics and Bioconductor infrastructure · 2015-06-23 · CSAMA 17 June 2015. Outline Proteomics and MS data Bioconductor infrastructure ... 5.0e+07 1.0e+08](https://reader034.vdocuments.net/reader034/viewer/2022042323/5f0dbd5d7e708231d43bd8f0/html5/thumbnails/23.jpg)
Ranges infrastructure
gs ge
G
T1
esi ee
i
i = 1
esi ee
i
i = 2
esi ee
i
i = 3
esi ee
i
i = 4
T2i = 1 i = 3 i = 4
T3i = 1 i = 4
P
psj pe
j
j = 1
psj pe
j
j = 2
psj pe
j
j = 3
Pj = 1 j = 2 j = 31 LP
Π
πsk πe
k
k = 1
πsk πe
k
k = 2
πskπe
k
k = 3
πskπe
k
k = 4
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Pbase package
library("Pbase")
p <- Proteins("uniprot.fasta")
p <- addIdentificationData(p, "identification.mzid")
aa(p) ## peptides sequences as a AAStringSet
pranges(p) ## peptide ranges as IRangesList
i <- which(acols(p)[, "EntryName"] == "EF2_HUMAN")
plot(p[i])
plot(p[i], from = 155, to = 185)
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Along protein coordinates
100
200
300
400
500
600
700
800
NH COOH
pept
ides
230
240
250NH COOH
P13
639
W A F T L K Q F A E M Y V A K F A A K G E G Q L G P A E R A K K V E
pept
ides
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Along genome coordinates
... using transcript models as GRangesList and Gviz for plotting.
Chromosome 1
156.11 mb
156.115 mb
156.12 mb
156.125 mb
156.13 mb
156.135 mb
pept
ides
SYLLGNSSPRTQSPQNCSIM
RATRSGAQASSTPLSPTR
SVGGSGGGSFGDNLVTR
RATRSGAQASSTPLSPTR ELEKTYSAKLDNAR
METPSQRRATR
DTSRRLLAEKEREMAEMR
LALDMEIHAYR
LALDMEIHAYRK
RQNGDDPLLTYRFPPK
SVGGSGGGSFGDNLVTR
SVGGSGGGSFGDNLVTR
From the Pbase mapping vignette.
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Along genome coordinates (with raw data)
Chromosome X
78.11 mb
78.115 mb
78.12 mb
78.125 mb
5' 3'3' 5'
MS
2 sp
ectr
aE
NS
T00
0003
7331
6
From the Pbase mapping vignette.
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With RNA-Seq readsChromosome 17
30.12 mb
30.13 mb
30.14 mb
30.15 mb
30.16 mb
30.17 mb
30.18 mb
EN
ST
0000
0612
959
0
20
40
60
80
Alig
nmen
tsTr
ack
From https://github.com/ComputationalProteomicsUnit/Intro-Integ-Omics-Prot
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Spatial proteomics
I The cellular sub-division
allows cells to establish a
range of distinct
microenvironments, each
favouring di�erent
biochemical reactions and
interactions and, therefore,
allowing each compartment
to ful�l a particular
functional role.
I Localisation and
sequestration of proteins
within subcellular niches is a
fundamental mechanism for
the post-translational
regulation of protein
function. Spatial proteomics is the systematic
study of protein localisations.
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Figure : Immuno�uorescence:ZFPL1, Golgi (left) and FHL2,mainly localized to actin �lamentsand focal adhesion sites. Alsodetected in the nucleus (right).(from the Human Protein Atlas)
Cell lysatefraction
centrifugation
iTRAQMS/MS
LOPIT(PCA,
PLS-DA)
label-freeMS/MS
(χ )2PCP
Pure fraction
catalogue
Invariantrich
fraction(clustering)
Subtractiveproteomics
(enrichment)
Figure : Mass spectrometry-basedapproaches based on densitygradient subcellular fractionation.
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Cell membrane lysis
Mechanical or bu�er-induced lysis of the plasma membrane with
minimal disruption to intracellular organelles followed by subcellular
fractionation.
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Density gradient separation
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Quantitation by LC-MSMS
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Data
Fraction1 Fraction2 . . . Fractionm markers
p1 q1,1 q1,2 . . . q1, m unknown
p2 q2,1 q2,2 . . . q2, m loc1
p3 q3,1 q3,2 . . . q3, m unknown
p4 q4,1 q4,2 . . . q4, m lock...
......
......
...
pn qn,1 qn,2 . . . qn, m unknown
Data analysis
MSnbase for data manipulation, pRoloc for clustering, classi�cation
and plotting, and pRolocGUI for interactive exploration.
![Page 35: Introduction to MS-based proteomics and Bioconductor infrastructure · 2015-06-23 · CSAMA 17 June 2015. Outline Proteomics and MS data Bioconductor infrastructure ... 5.0e+07 1.0e+08](https://reader034.vdocuments.net/reader034/viewer/2022042323/5f0dbd5d7e708231d43bd8f0/html5/thumbnails/35.jpg)
0.2
0.3
0.4
0.5
Correlation profile − ER
Fractions
1 2 4 5 7 81112
0.1
0.2
0.3
0.4
Correlation profile − Golgi
Fractions
1 2 4 5 7 81112
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Correlation profile − mit/plastid
Fractions
1 2 4 5 7 81112
0.15
0.20
0.25
0.30
0.35
Correlation profile − PM
Fractions
1 2 4 5 7 81112
0.1
0.2
0.3
0.4
0.5
0.6
Correlation profile − Vacuole
Fractions
1 2 4 5 7 81112
●●
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−10 −5 0 5
−5
05
Principal component analysis
PC1
PC
2
●
ERGolgimit/plastidPM
vacuolemarkerPLS−DAunknown
Figure : From Gatto et al. (2010), data from Dunkley et al. (2006).
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2009 vs 2013
−3 −2 −1 0 1 2 3
−3
−2
−1
01
23
PC1 (58.53%)
PC
2 (2
9.96
%)
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ER/GolgimitochondrionPMunknown
−3 −2 −1 0 1 2 3
−3
−2
−1
01
23
PC1 (58.53%)
PC
2 (2
9.96
%)
CytoskeletonERGolgiLysosomemitochondrionNucleus
PeroxisomePMProteasomeRibosome 40SRibosome 60S
Figure : Semi-supervised approach Breckels et al. (2013). Data from Tanet al (2009).
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−6 −4 −2 0 2 4
−4
−2
02
4
PC1 (50.05%)
PC
2 (2
4.61
%)
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Actin cytoskeletonCytosolEndosomeER/GAExtracellular matrixLysosomeMitochondriaNucleus − ChromatinNucleus − NucleolusPeroxisomePlasma MembraneProteasomeRibosome 40SRibosome 60Sunknown
●●●●●●
111111
●●●●●●222222 ●
●●●3333
●●●●●
●●●4 4
4444
44
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55
5
55
5 5
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● 66 666
6 6
6
6
●●●●●●●●●
● ●● 777 7 77 7
777 7
7
●●●●● ●●●
88888 8
88
●●●● ●●●
●
9999 999
9
1 Dynein2 Vesicles − Clathrin 3 13S condensin4 T complex5 Nucleus lamina
6 Vesicles − COPI/II7 eIF3 complex8 ARP2/3 complex9 COP9 signalosome
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
![Page 38: Introduction to MS-based proteomics and Bioconductor infrastructure · 2015-06-23 · CSAMA 17 June 2015. Outline Proteomics and MS data Bioconductor infrastructure ... 5.0e+07 1.0e+08](https://reader034.vdocuments.net/reader034/viewer/2022042323/5f0dbd5d7e708231d43bd8f0/html5/thumbnails/38.jpg)
Acknowledgement
I Lisa Breckels
I Sebastien Gibb
I Kathryn Lilley
(CCP)
## R version 3.2.0 Patched (2015-04-22 r68234)## Platform: x86_64-unknown-linux-gnu (64-bit)## Running under: Ubuntu 14.04.2 LTS#### attached base packages:## [1] stats graphics grDevices utils datasets methods base#### loaded via a namespace (and not attached):## [1] Biobase_2.29.1 vsn_3.37.1## [3] splines_3.2.0 foreach_1.4.2## [5] Formula_1.2-1 affy_1.47.1## [7] Pbase_0.9.0 highr_0.5## [9] stats4_3.2.0 latticeExtra_0.6-26## [11] BSgenome_1.37.1 Rsamtools_1.21.8## [13] impute_1.43.0 RSQLite_1.0.0## [15] lattice_0.20-31 biovizBase_1.17.1## [17] limma_3.25.9 chron_2.3-45## [19] digest_0.6.8 GenomicRanges_1.21.15## [21] RColorBrewer_1.1-2 XVector_0.9.1## [23] colorspace_1.2-6 preprocessCore_1.31.0## [25] plyr_1.8.2 MALDIquant_1.12## [27] XML_3.98-1.2 biomaRt_2.25.1## [29] zlibbioc_1.15.0 scales_0.2.4## [31] affyio_1.37.0 cleaver_1.7.0## [33] BiocParallel_1.3.25 IRanges_2.3.11## [35] ggplot2_1.0.1 SummarizedExperiment_0.1.5## [37] GenomicFeatures_1.21.13 nnet_7.3-9## [39] Gviz_1.13.2 BiocGenerics_0.15.2## [41] proto_0.3-10 survival_2.38-1## [43] magrittr_1.5 evaluate_0.7## [45] doParallel_1.0.8 MASS_7.3-40## [47] foreign_0.8-63 mzR_2.3.1## [49] BiocInstaller_1.19.6 Pviz_1.3.0## [51] tools_3.2.0 data.table_1.9.4## [53] formatR_1.2 matrixStats_0.14.0## [55] stringr_1.0.0 MSnbase_1.17.5## [57] S4Vectors_0.7.5 munsell_0.4.2## [59] cluster_2.0.1 AnnotationDbi_1.31.16## [61] lambda.r_1.1.7 Biostrings_2.37.2## [63] pcaMethods_1.59.0 GenomeInfoDb_1.5.7## [65] mzID_1.7.0 futile.logger_1.4.1## [67] grid_3.2.0 RCurl_1.95-4.6## [69] dichromat_2.0-0 iterators_1.0.7## [71] VariantAnnotation_1.15.13 bitops_1.0-6## [73] gtable_0.1.2 codetools_0.2-11## [75] DBI_0.3.1 reshape2_1.4.1## [77] GenomicAlignments_1.5.9 gridExtra_0.9.1## [79] knitr_1.10.5 rtracklayer_1.29.10## [81] Hmisc_3.16-0 ProtGenerics_1.1.0## [83] futile.options_1.0.0 stringi_0.4-1## [85] parallel_3.2.0 Rcpp_0.11.6## [87] rpart_4.1-9 acepack_1.3-3.3