SubCellBarCode: Integrated workflow for robust classification and visualization of spatial proteome
2024-10-29
Abstract
In the SubCellBarCode project, mass spectrometry (MS) based proteomics was used for proteome-wide mapping of protein subcellular localization (Orre et al. 2019, Molecular Cell). Subcellular fractionation and MS-based quantification can be reproduced following the methods described in Orre et al. Here, the SubCellBarCode R package offers tools to reproduce the underlying bioinformatics pipeline for robust classification and visualization of proteins into corresponding subcellular localizations. Moreover, the R-package can be used for differential localization analysis to investigate e.g. treatment induced protein relocalization, condition dependent localization or cell type specific localization. Last but not least, it provides peptide/ exon/transcript centric classification as well as PTM (Post translation modifications) modified peptide classification.Package
SubCellBarCode 1.23.0
Contents
- 1 Installation of the package
- 2 Load the package
- 3 Data preparation and classification
- 3.1 Example Data
- 3.2 Marker Proteins
- 3.3 Load and normalize data
- 3.4 Calculate covered marker proteins
- 3.5 Quality control of the marker proteins
- 3.6 Visualization of marker proteins in t-SNE map
- 3.7 Build model and classify proteins
- 3.7.1 Estimate classification thresholds for compartment level
- 3.7.2 Apply threshold to compartment level classifications
- 3.7.3 Estimate classification thresholds for neighborhood level
- 3.7.4 Apply threshold to neighborhood level classifications
- 3.7.5 Merge compartment and neighborhood classification
- 4 Visualization of the protein subcellular localization
- 5 Differential localization analysis
- 6 Peptide/Exon/Transcript centric or PTM regulated localization
- 7 References
- Appendix
- A Session Information
1 Installation of the package
SubCellBarCode can be installed through BiocManager package as follows:
if (!requireNamespace("BiocManager"))
install.packages("BiocManager")
BiocManager::install("SubCellBarCode")2 Load the package
library(SubCellBarCode)## 3 Data preparation and classification
3.1 Example Data
As example data we here provide the publicly available HCC827 (human lung
adenocarcinoma cell line) TMT10plex labelled proteomics dataset
(Orre et al. 2019, Molecular Cell). The data.frame consists of
10480 proteins as rows (rownames are gene -centric protein ids)
and 5 fractions with duplicates as columns
(replicates must be named “.A.” and “.B.”, repectively).
head(hcc827Ctrl)## FS1.A.HCC827 FS1.B.HCC827 FS2.A.HCC827 FS2.B.HCC827 FP1.A.HCC827
## A2M 6.6567 4.8238 0.8265 0.8279 0.4475
## A2ML1 1.2876 1.0878 0.6390 0.7828 0.8760
## A4GALT 0.4711 0.4106 0.2742 0.2689 0.8389
## AAAS 0.5108 0.4514 0.4470 0.4752 1.6576
## AACS 4.5593 4.4522 1.5694 1.6417 0.5294
## AAED1 0.8170 0.7031 0.5415 0.5902 0.9429
## FP1.B.HCC827 FP2.A.HCC827 FP2.B.HCC827 FP3.A.HCC827 FP3.B.HCC827
## A2M 0.5803 0.6414 0.6014 0.6497 0.6216
## A2ML1 0.9138 2.6957 1.3606 0.7715 0.7859
## A4GALT 0.8043 1.9637 1.8340 1.6739 1.6848
## AAAS 1.7316 1.3773 1.4688 1.0071 1.0457
## AACS 0.5197 0.5109 0.5232 0.4197 0.4243
## AAED1 0.9505 1.9035 1.8475 1.1060 1.16363.2 Marker Proteins
The classification of protein localisation using the SubCellBarCode
method is dependent on 3365 marker proteins as defined in Orre et al.
The markerProteins data.frame contain protein names (gene symbol),
associated subcellular localization (compartment), color code for
the compartment and the median normalized fractionation
profile (log2) based on five different human cell lines
(NCI-H322, HCC827, MCF7, A431and U251) here called the
“5CL marker profile”.
head(markerProteins)## Proteins Compartments Cyto Nsol NucI Horg
## AAAS AAAS S4 -1.0033518 -1.15489468 0.9303367 0.48554266
## AACS AACS C4 2.1716569 0.13246046 -1.0394634 -1.13265585
## AAK1 AAK1 C3 1.8556445 0.10015281 -0.6605511 -0.36985132
## AARS AARS C4 2.1012831 0.05855811 -1.0439250 -1.08451971
## AASDHPPT AASDHPPT C5 1.7065897 0.51507061 -0.7196594 -0.65267201
## AATF AATF N3 -0.8922053 -1.10101864 1.2202070 0.05316807
## Lorg Colour
## AAAS 0.1618329 tomato2
## AACS -1.3217619 deepskyblue2
## AAK1 -0.6741959 cyan
## AARS -1.2180979 deepskyblue2
## AASDHPPT -1.0059841 turquoise3
## AATF 0.1898676 grey503.3 Load and normalize data
Input data.frame is checked with “NA” values and for the correct
format. If there is any “NA” value, corresponding row is deleted.
Then, data frame is log2 transformmed.
df <- loadData(protein.data = hcc827Ctrl)cat(dim(df))## 10480 10head(df)## FS1.A.HCC827 FS1.B.HCC827 FS2.A.HCC827 FS2.B.HCC827 FP1.A.HCC827
## A2M 2.7348072 2.2701701 -0.2749133 -0.2724716 -1.16004041
## A2ML1 0.3646845 0.1214133 -0.6461122 -0.3532843 -0.19099723
## A4GALT -1.0858948 -1.2841945 -1.8666995 -1.8948583 -0.25342925
## AAAS -0.9691696 -1.1475217 -1.1616533 -1.0733933 0.72909591
## AACS 2.1888123 2.1545184 0.6502131 0.7151905 -0.91756990
## AAED1 -0.2915920 -0.5081982 -0.8849668 -0.7607242 -0.08482332
## FP1.B.HCC827 FP2.A.HCC827 FP2.B.HCC827 FP3.A.HCC827 FP3.B.HCC827
## A2M -0.78512917 -0.6407037 -0.7336032 -0.62215439 -0.68594159
## A2ML1 -0.13004965 1.4306600 0.4442430 -0.37426194 -0.34758234
## A4GALT -0.31419437 0.9735745 0.8749936 0.74321334 0.75257734
## AAAS 0.79210571 0.4618428 0.5546380 0.01020694 0.06446902
## AACS -0.94424904 -0.9688872 -0.9345656 -1.25256963 -1.23684342
## AAED1 -0.07324147 0.9286546 0.8855744 0.14535139 0.21859520Additional step:
We use gene symbols for the protein identification. Therefore, we require
gene symbols for the identifiaction. However, if the input data has
other identifier e.g. UNIPROT, IPI, Entrez ID, you can convert it to gene
symbol by our defined function.
Please be aware of possible (most likely few) id loss during the
conversion to one another.
##Run if you have another identifier than gene symbols.
##Function will convert UNIPROT identifier to gene symbols.
##Deafult id is "UNIPROT", make sure you change it if you use another.
#df <- convert2symbol(df = df, id = "UNIPROT")For the downstream analysis, we used the randomly selected subset data.
set.seed(2)
df <- df[sample(nrow(df), 6000),]3.4 Calculate covered marker proteins
The overlap between marker proteins (3365) and input data.frame is calculated and visualized for each compartment by a bar plot.
Note that we recommend at least 20% coverage of marker proteins for each compartment. If certain compartments are underrrepresented we recommend you to perform the cell fractionation again. If all compartments are low in coverage we recommend increasing the analytical depth of the MS-analysis.
c.prots <- calculateCoveredProtein(proteinIDs = rownames(df),
markerproteins = markerProteins[,1]) 
## Overall Coverage of marker proteins : 0.583.5 Quality control of the marker proteins
To avoid reduced classification accuracy, marker proteins with noisy quantification and marker proteins that are not representative of their associated compartment (e.g.due to cell type specific localization) are filtered out by a two-step quality control.
Marker proteins with pearson correlations less than 0.8 between A and B duplicates for each cell line were filtered out (Figure A).
Pairwise correlations between 5CL marker profile and input data for each protein (A and B replicate experiments separately) were calculated using both Pearson and Spearman correlation. The lowest value for each method were then used for filtering with cut-offs set to 0.8 and 0.6 respectively, to exclude non-representative marker proteins (Figure B).
r.markers <- markerQualityControl(coveredProteins = c.prots,protein.data = df)## Number of removed replicate-wise proteins: 0## Number of removed sample-wise proteins: 1## Number of total removed marker proteins: 1
r.markers[1:5]## [1] "N4BP2L2" "FURIN" "ATG9A" "ZNF205" "MAVS"Optional step: After removing non-marker proteins, you can re-calculate and visualize the final coverage of the marker proteins.
## uncomment the function when running
# f.prots <- calculateCoveredProtein(r.markers, markerProteins[,1])3.6 Visualization of marker proteins in t-SNE map
The spatial distribution of the marker proteins is visualized in t-SNE map. This plot will be informative for the quality control of the generated data as it offers evaluation of the spatial distribution and separation of marker proteins.
#Default parameters
#Run the broad-range parameters to optimize well !!!
#Output dimensionality
#dims = 3
#Speed/accuracy trade-off (increase for less accuracy)
#theta = c(0.1, 0.2, 0.3, 0.4, 0.5)
#Perplexity parameter
#perplexity = c(5, 10, 20, 30, 40, 50, 60) Information about the different t-SNE parameters that can be
modified by the user is available by typing ?Rtsne in the console.
Although the applications of t-SNE is widespread in the field
of machine learning, it can be misleading if it is not well
optimized. Therefore, we optimize t-SNE map by grid search,
a process that can take some time
set.seed(6)
tsne.map <- tsneVisualization(protein.data = df,
markerProteins = r.markers,
dims = 3,
theta = c(0.1),
perplexity = c(60)) ## Optimization process started. This may take some time!## Optimization was performed.## Theta value: 0.1
## Perplexity value: 60
We recommend 3D vizualisation by setting dims = 3,
for optimal evaluation of marker protein cluster separation
and data modularity. You can also visualize the marker
proteins in 2 dimensional space by setting dims = 2, although
reducing the dimensionality results in loss of information and
underestimation of data resolution.
set.seed(9)
tsne.map2 <- tsneVisualization(protein.data = df,
markerProteins = r.markers,
dims = 2,
theta = c(0.5),
perplexity = c(60))## Optimization process started. This may take some time!## Optimization was performed.## Theta value: 0.5
## Perplexity value: 60
3.7 Build model and classify proteins
For replicate A and B separately, marker proteins are used for training a Support Vector Machine (SVM) classifier with a Gaussian radial basis function kernel algorithm. After tuning the parameters, the SVM model predicts (classifies) the subcellular localization for all proteins in the input data with corresponding probabilities for A and B replicate classification.
set.seed(4)
cls <- svmClassification(markerProteins = r.markers,
protein.data = df,
markerprot.df = markerProteins)# testing data predictions for replicate A and B
test.A <- cls[[1]]$svm.test.prob.out
test.B <- cls[[2]]$svm.test.prob.out
head(test.A)## Observation svm.pred S1 S2 S3 S4
## N4BP2L2 N2 N2 0.0034204982 0.002713603 0.003135572 0.002424749
## ZNF205 N1 N1 0.0539341925 0.005611837 0.009373929 0.003251983
## MAVS M2 M2 0.0005904669 0.001188552 0.001075943 0.001988799
## NGLY1 C2 C2 0.0020717253 0.001835965 0.001706640 0.001777443
## RAB15 S2 S2 0.1005330801 0.850062711 0.017526771 0.022986723
## CHD4 N2 N2 0.0093310932 0.009050335 0.009640417 0.007007189
## N1 N2 N3 N4 C1
## N4BP2L2 0.0370000247 0.8956709656 0.026904787 0.0127955674 0.0026575959
## ZNF205 0.9047763896 0.0040535626 0.005468579 0.0024351136 0.0022360891
## MAVS 0.0009300487 0.0010955494 0.001556564 0.0010128144 0.0008742056
## NGLY1 0.0021911577 0.0017599204 0.001974421 0.0090865761 0.0608829293
## RAB15 0.0006195267 0.0006601397 0.000699539 0.0009793257 0.0009498403
## CHD4 0.0147735324 0.5904820545 0.016469668 0.3054409175 0.0060342981
## C2 C3 C4 C5 M1
## N4BP2L2 0.0020172675 0.0019633826 0.0022083395 0.0027406564 0.0026281681
## ZNF205 0.0014451171 0.0011720556 0.0013531775 0.0016422573 0.0016849212
## MAVS 0.0008075442 0.0006829592 0.0008801080 0.0010014552 0.0341655989
## NGLY1 0.8509162130 0.0393558124 0.0031946915 0.0198804447 0.0016605889
## RAB15 0.0008681537 0.0007352767 0.0007446856 0.0008892102 0.0009869426
## CHD4 0.0044846791 0.0035101058 0.0037620803 0.0055981694 0.0090452900
## M2
## N4BP2L2 0.0017188220
## ZNF205 0.0015607954
## MAVS 0.9521493909
## NGLY1 0.0017054720
## RAB15 0.0007580757
## CHD4 0.0053701723# all predictions for replicate A and B
all.A <- cls[[1]]$all.prot.pred
all.B <- cls[[2]]$all.prot.pred3.7.1 Estimate classification thresholds for compartment level
Classification probabilities close to 1 indicate high confidence predictions, whereas probabilities close to 0 indicate low confidence predictions. To increase the overal prediction accuracy and to filter out poor predictions, one criterion and two cut-offs are defined.
The criterion is the consensus of preliminary predictions between biological duplicates. Proteins are kept in the analysis, if there is an agreement between biological duplicates. Subsequently, prediction probabilities from the two duplicates are averaged for each protein.
Cut-off is (precision - based) set when precision reach 0.9 in the test data.
Cut-off is (recall - based) set as the probability of the lowest true positive in the test data.
t.c.df <- computeThresholdCompartment(test.repA = test.A, test.repB = test.B)head(t.c.df)## Compartment PrecisionBasedThreshold RecallBasedThreshold OptedThreshold
## 1 S1 0.47 0.495 0.495
## 2 S2 0.785 0.51 0.785
## 3 S3 0.535 0.375 0.535
## 4 S4 0.68 0.445 0.680
## 5 N1 0 0.72 0.720
## 6 N2 0 0.53 0.5303.7.2 Apply threshold to compartment level classifications
The determined thresholds for the compartment levels are applied to all classifications.
c.cls.df <- applyThresholdCompartment(all.repA = all.A, all.repB = all.B,
threshold.df = t.c.df)head(c.cls.df)## Proteins svm.pred S1 S2 S3 S4
## FURIN FURIN S1 0.8044625 0.118285221 0.06148853 0.0039862411
## ATG9A ATG9A S1 0.8482675 0.013538605 0.01131488 0.0042270023
## DISC1 DISC1 S1 0.6003724 0.034584719 0.13175231 0.0231587190
## HVCN1 HVCN1 S1 0.9382960 0.005664071 0.01047046 0.0019191809
## VTI1A VTI1A S1 0.9802552 0.002545447 0.01070461 0.0004074506
## SLC35F2 SLC35F2 S1 0.7683913 0.019822117 0.16210238 0.0081724862
## N1 N2 N3 N4 C1
## FURIN 0.001063479 0.0011090607 0.0008779197 0.0010555088 0.0009208802
## ATG9A 0.085590105 0.0032762068 0.0045218381 0.0053467703 0.0066286150
## DISC1 0.045068474 0.0075255478 0.0069405674 0.0109770978 0.0939001191
## HVCN1 0.032281961 0.0013498275 0.0013276950 0.0014380712 0.0015681802
## VTI1A 0.001194382 0.0005728802 0.0005238379 0.0005018539 0.0005017726
## SLC35F2 0.017392373 0.0046300609 0.0025411416 0.0030319662 0.0041498757
## C2 C3 C4 C5 M1
## FURIN 0.0008966154 0.0007727800 0.0007533706 0.0009464182 0.0019590025
## ATG9A 0.0034716395 0.0024226870 0.0020141834 0.0035120378 0.0027487307
## DISC1 0.0175978565 0.0071947713 0.0035340674 0.0065186738 0.0044177332
## HVCN1 0.0010475977 0.0008637610 0.0008279222 0.0010973763 0.0008732059
## VTI1A 0.0005178166 0.0004366861 0.0004628942 0.0005308447 0.0004443072
## SLC35F2 0.0019535196 0.0013716268 0.0012046753 0.0017430045 0.0016115742
## M2
## FURIN 0.0014224819
## ATG9A 0.0031192271
## DISC1 0.0064569012
## HVCN1 0.0009747184
## VTI1A 0.0004000427
## SLC35F2 0.00188195013.7.3 Estimate classification thresholds for neighborhood level
Compartment level classification probabilities are summed to neighborhood probabilities and thresholds for neighborhood analysis are estimated as described above for compartment level analysis except precision based cut-off is set to 0.95.
t.n.df <- computeThresholdNeighborhood(test.repA = test.A, test.repB = test.B)head(t.n.df)## Neighborhood PrecisionBasedThreshold RecallBasedThreshold OptedThreshold
## 1 Secretory 0 0.65 0.650
## 2 Nuclear 0 0.47 0.470
## 3 Cytosol 0 0.58 0.580
## 4 Mitochondria 0 0.655 0.6553.7.4 Apply threshold to neighborhood level classifications
The determined thresholds for the neighborhood levels are applied to all classifications.
n.cls.df <- applyThresholdNeighborhood(all.repA = all.A, all.repB = all.B,
threshold.df = t.n.df)head(n.cls.df)## Proteins svm.pred.all Secretory Nuclear Cytosol Mitochondria
## FURIN FURIN Secretory 0.9882225 0.004105968 0.004290065 0.003381484
## ATG9A ATG9A Secretory 0.8773480 0.098734920 0.018049163 0.005867958
## DISC1 DISC1 Secretory 0.7898682 0.070511687 0.128745488 0.010874634
## HVCN1 HVCN1 Secretory 0.9563497 0.036397555 0.005404837 0.001847924
## VTI1A VTI1A Secretory 0.9939127 0.002792954 0.002450014 0.000844350
## CEP68 CEP68 Secretory 0.9815079 0.006802636 0.007935830 0.0037536593.7.5 Merge compartment and neighborhood classification
Individual classifications (compartment and neighborhood) are merged into one data frame.
cls.df <- mergeCls(compartmentCls = c.cls.df, neighborhoodCls = n.cls.df)head(cls.df)## Protein NeighborhoodCls CompartmentCls Secretory Nuclear
## A2M A2M Cytosol Unclassified 0.108974693 0.148019019
## A2ML1 A2ML1 Unclassified Unclassified 0.289312766 0.106674311
## A4GALT A4GALT Secretory S1 0.984063133 0.006326616
## AACS AACS Cytosol Unclassified 0.007062643 0.009852283
## AAGAB AAGAB Cytosol Unclassified 0.006046029 0.006551317
## AAK1 AAK1 Cytosol Unclassified 0.005356143 0.006359080
## Cytosol Mitochondria S1 S2 S3 S4
## A2M 0.707476254 0.035530034 0.039866660 0.035645043 0.019382476 0.014080514
## A2ML1 0.313939713 0.290073210 0.068059420 0.052162470 0.104065175 0.065025702
## A4GALT 0.007018236 0.002592015 0.967094493 0.013246951 0.002326302 0.001395387
## AACS 0.979996757 0.003088316 0.002163661 0.001971189 0.001603501 0.001324293
## AAGAB 0.984621943 0.002780711 0.001702959 0.001659116 0.001446875 0.001237080
## AAK1 0.985783207 0.002501570 0.001509276 0.001434766 0.001288690 0.001123411
## N1 N2 N3 N4 C1 C2
## A2M 0.043316074 0.018332245 0.024952969 0.061417730 0.173374778 0.043123461
## A2ML1 0.028041362 0.013005800 0.017333591 0.048293558 0.107401033 0.127785177
## A4GALT 0.001279695 0.001378964 0.001444322 0.002223634 0.001873890 0.001430963
## AACS 0.002292800 0.001597228 0.001781031 0.004181224 0.004083417 0.002940375
## AAGAB 0.001966653 0.001460906 0.001589922 0.001533835 0.490438977 0.018607661
## AAK1 0.001677854 0.001288555 0.001389773 0.002002898 0.367893604 0.018527206
## C3 C4 C5 M1 M2
## A2M 0.120829788 0.209961620 0.160186606 0.013156336 0.022373699
## A2ML1 0.045365976 0.011057367 0.022330159 0.025059542 0.265013668
## A4GALT 0.001156778 0.001033019 0.001523587 0.001447960 0.001144055
## AACS 0.016174572 0.580101553 0.376696841 0.001440217 0.001648100
## AAGAB 0.444608775 0.009851353 0.021115177 0.001371630 0.001409080
## AAK1 0.557600944 0.011618468 0.030142985 0.001246429 0.0012551424 Visualization of the protein subcellular localization
4.1 SubCellBarCode plot
You can query one protein at a time to plot barcode of the protein of the interest.
PSM (Peptide-spectra-matching) count table is required for the plotting
SubCellBarCode. It is in data.frame format;
head(hcc827CtrlPSMCount)## Protein PSMs.for.quant
## A2M A2M 11
## A2ML1 A2ML1 2
## A4GALT A4GALT 1
## AAAS AAAS 33
## AACS AACS 60
## AAED1 AAED1 9plotBarcode(sampleClassification = cls.df, protein = "NLRP4",
s1PSM = hcc827CtrlPSMCount)
4.2 Co-localization plot
To evaluate localization and of multiple proteins at the same time, a vector of proteins (identified by gene symbols) can be prepared and used to create a barplot showing the distribution of classifications across compartments and neighborhoods. This analysis could be helpful when evaluating co-localization of proteins, protein complex formation and compartmentalized protein level regulation.
# 26S proteasome complex (26s proteasome regulatory complex)
proteasome26s <- c("PSMA7", "PSMC3","PSMA4", "PSMB4",
"PSMB6", "PSMB5", "PSMC2","PSMC4",
"PSMB3", "PSMA6","PSMC5","PSMC6")
plotMultipleProtein(sampleClassification = cls.df, proteinList = proteasome26s)## PSMB4,PSMC6 not exist in data
5 Differential localization analysis
Regulation of protein localization is a the key
process in cellular signalling. The SubCellBarCode
method can be used for differential localization analysis
given two conditions such as control vs treatment, cancer
cell vs normal cell, cell state A vs cell state B, etc.
As example, we compared untreated and gefitinib (EGFR inhibitor)
treated HCC827 cells (for details, see Orre et al.).
5.1 Plot differentially localizing proteins
Neighborhood classifications for condition 1 (untreated) and
condition 2 (gefitinib) is first done separately, and
classifications for overlapping proteins are then vizualized by a
sankey plot.
The HCC827 gefitinib cell lines classification was embedded
into the package for example analysis.
head(hcc827GEFClass)## Protein NeighborhoodCls CompartmentCls
## A2M A2M Cytosol Unclassified
## A2ML1 A2ML1 Unclassified Unclassified
## A4GALT A4GALT Secretory S1
## AAAS AAAS Secretory S4
## AACS AACS Cytosol Unclassified
## AAED1 AAED1 Secretory UnclassifiedsankeyPlot(sampleCls1 = cls.df, sampleCls2 = hcc827GEFClass)## Cond1 Cond2 value
## 1 Secretory Secretory 1323
## 2 Secretory Nuclear 19
## 3 Secretory Cytosol 14
## 4 Secretory Mitochondria 9
## 5 Nuclear Secretory 15
## 6 Nuclear Nuclear 1263
## 7 Nuclear Cytosol 23
## 8 Nuclear Mitochondria 0
## 9 Cytosol Secretory 39
## 10 Cytosol Nuclear 36
## 11 Cytosol Cytosol 2019
## 12 Cytosol Mitochondria 1
## 13 Mitochondria Secretory 5
## 14 Mitochondria Nuclear 1
## 15 Mitochondria Cytosol 1
## 16 Mitochondria Mitochondria 3345.2 Filter Candidates
As the differential localization analysis is an outlier analysis,
it will include analytical noise. To filter out such noise,
PSM (Peptide-spectra-matching) counts and fractionation profile
correlation analysis (Pearson) was done to identify strong
candidates. The PSM count format for the input have to be the
same between the compared conditions;
head(hcc827CtrlPSMCount)## Protein PSMs.for.quant
## A2M A2M 11
## A2ML1 A2ML1 2
## A4GALT A4GALT 1
## AAAS AAAS 33
## AACS AACS 60
## AAED1 AAED1 9For each protein, the minimum PSM count between the two conditions
is plotted against the fractionation profile (median) correlation
between the two conditions. For proteins with different localizations
between conditions, the fractionation profile differs and therefore
we are expecting a low fractionation profile correlation. As a standard
setting for filtering of analytical noise in the differential
localization analysis we suggest to demand a fractionation profile
correlation below 0.8, and a minimum PSM count of at least 3.
However, for the exploratory analysis, you can adjust
the min.psm and pearson.cor parameters.
##parameters
#sampleCls1 = sample 1 classification output
#s1PSM = sample 2 PSM count
#s1Quant = Sample 1 Quantification data
#sampleCls2 = sample 2 classification output
#s2PSM = sample 2 classification output
#sample2Quant = Sample 2 Quantification data
#min.psm = minumum psm count
#pearson.cor = perason correlation coefficientcandidate.df <- candidateRelocatedProteins(sampleCls1 = cls.df,
s1PSM = hcc827CtrlPSMCount,
s1Quant = hcc827Ctrl,
sampleCls2 = hcc827GEFClass,
s2PSM = hcc827GefPSMCount,
s2Quant = hcc827GEF,
min.psm = 2,
pearson.cor = 0.8)## Warning: Use of `f.df$Min.PSMs` is discouraged.
## ℹ Use `Min.PSMs` instead.
cat(dim(candidate.df))## 35 5head(candidate.df)## Protein C.A C.B Pearson.Corr Min.PSMs
## ACD ACD Cytosol Nuclear 0.7274418 4
## AGAP2 AGAP2 Nuclear Cytosol 0.4888424 3
## ANKEF1 ANKEF1 Cytosol Secretory 0.5230030 3
## BATF3 BATF3 Secretory Cytosol -0.6132878 5
## CGNL1 CGNL1 Cytosol Nuclear 0.7850309 34
## CRY1 CRY1 Nuclear Secretory -0.7397292 4Candidate subset of differentially localizing proteins can
be annotated with names by setting annotation = TRUE, min.psm
and pearson.cor
candidate2.df <- candidateRelocatedProteins(sampleCls1 = cls.df,
s1PSM = hcc827CtrlPSMCount,
s1Quant = hcc827Ctrl,
sampleCls2 = hcc827GEFClass,
s2PSM = hcc827GefPSMCount,
s2Quant = hcc827GEF,
annotation = TRUE,
min.psm = 9,
pearson.cor = 0.05) ## Warning: Use of `annot.df$Min.PSMs` is discouraged.
## ℹ Use `Min.PSMs` instead.## Warning: Use of `annot.df$Label` is discouraged.
## ℹ Use `Label` instead.## Warning: Use of `annot.df$Min.PSMs` is discouraged.
## ℹ Use `Min.PSMs` instead.## Warning: Use of `annot.df$Label` is discouraged.
## ℹ Use `Label` instead.
6 Peptide/Exon/Transcript centric or PTM regulated localization
Our main analysis is based on gene-centric. However, based on the analytical
depth of the data, peptide/exon/transcript centric classifications can
be performed. Moreover, this approach is also applicable for
post translation modification (PTM) enriched data.
Fundamentally, we will use the same classifiers, compartment
and neighborhood levels thresholds and apply
them to peptide/exon/transcript data.
6.1 Exon-centric classification
Here, we have provided subset of the HCC827 exon-centric data.
head(hcc827exon)## Gene_Symbol FS1.A.HCC827 FS1.B.HCC827 FS2.A.HCC827 FS2.B.HCC827
## ENSE00000331191 CDC45 3.0054124 3.1763997 1.5782291 1.6024560
## ENSE00000331854 COPB1 2.0449415 2.0610331 1.1743850 1.1716880
## ENSE00000331855 COPB1 1.8797670 1.5457263 1.0152127 1.0173337
## ENSE00000331859 COPB1 1.7921392 2.0895850 1.0261406 1.0273679
## ENSE00000331990 LPIN1 2.4724838 2.7718197 1.4581675 1.7319290
## ENSE00000332881 PTPRA 0.4461752 0.6117839 0.5241694 0.3844395
## FP1.A.HCC827 FP1.B.HCC827 FP2.A.HCC827 FP2.B.HCC827
## ENSE00000331191 0.9467013 0.9600072 0.5214538 0.6500854
## ENSE00000331854 0.6919514 0.7403220 1.0573085 1.0332836
## ENSE00000331855 0.8060150 0.8152469 1.2035301 1.1714791
## ENSE00000331859 0.7443903 0.8078896 1.2703089 1.1974385
## ENSE00000331990 0.6075537 0.7533756 0.8890101 0.9232132
## ENSE00000332881 1.3494955 1.2581107 1.7331063 1.6535322
## FP3.A.HCC827 FP3.B.HCC827 PeptideCount
## ENSE00000331191 0.6017849 0.6252090 1
## ENSE00000331854 1.1354910 1.1858233 5
## ENSE00000331855 1.2002348 1.1099946 1
## ENSE00000331859 1.1113409 1.0545062 1
## ENSE00000331990 0.7477472 0.8117479 1
## ENSE00000332881 1.0713874 1.2001535 3For the classification, we will use the gene-centric model that we built in section 3.7.
##recall the models
modelA <- cls[[1]]$model
modelB <- cls[[2]]$modelsvmExternalData function greps replicates A and B, repectively. So
the input data can include other features like, here, peptide counts.
exon.cls <- svmExternalData(df = hcc827exon, modelA = modelA, modelB= modelB)exon.A <- exon.cls[[1]]
exon.B <- exon.cls[[2]]head(exon.A)## Gene_Symbol svm.pred.all S1 S2 S3
## ENSE00000331191 CDC45 C5 0.005209818 0.004428667 0.003555885
## ENSE00000331854 COPB1 C1 0.002094528 0.001549502 0.001638616
## ENSE00000331855 COPB1 C1 0.004767946 0.002673107 0.004305875
## ENSE00000331859 COPB1 C2 0.005863040 0.003269242 0.005165852
## ENSE00000331990 LPIN1 C1 0.001952427 0.001807642 0.001534898
## ENSE00000332881 PTPRA S4 0.005234257 0.041896085 0.382199347
## S4 N1 N2 N3 N4
## ENSE00000331191 0.003242003 0.005011824 0.003435750 0.004037131 0.014660840
## ENSE00000331854 0.001500378 0.002987032 0.001428675 0.001654051 0.003863658
## ENSE00000331855 0.003354011 0.004539758 0.001967439 0.002153011 0.006044831
## ENSE00000331859 0.003993647 0.005077045 0.002213735 0.002507557 0.008619431
## ENSE00000331990 0.001507256 0.002226228 0.001618174 0.001889669 0.008721204
## ENSE00000332881 0.431742588 0.002736390 0.007376895 0.003353097 0.002177273
## C1 C2 C3 C4 C5
## ENSE00000331191 0.029431200 0.02739400 0.104285450 0.079949261 0.708536210
## ENSE00000331854 0.545978939 0.38805113 0.041528864 0.002229591 0.002992308
## ENSE00000331855 0.512303625 0.38701929 0.059858677 0.002931555 0.004412370
## ENSE00000331859 0.394845949 0.48590743 0.069114609 0.003397616 0.005398430
## ENSE00000331990 0.495706311 0.20133219 0.223907357 0.010549736 0.044135377
## ENSE00000332881 0.001670115 0.00159227 0.001496738 0.001614183 0.001821697
## M1 M2
## ENSE00000331191 0.002993919 0.003828040
## ENSE00000331854 0.001215528 0.001287200
## ENSE00000331855 0.001570441 0.002098061
## ENSE00000331859 0.001887038 0.002739378
## ENSE00000331990 0.001460832 0.001650700
## ENSE00000332881 0.071655946 0.043433117Next steps are exactly same as what we did in section 3.7. We will use same thresholdsthat we have estimated in 3.7.1 and 3.7.3. Finally, we will merge two classifications same function used in 3.7.5.
exon.comp.cls <- applyThresholdCompartment(all.repA = exon.A[,2:17],
all.repB = exon.B[,2:17],
threshold.df = t.c.df)
exon.neigh.cls <- applyThresholdNeighborhood(all.repA = exon.A[,2:17],
all.repB = exon.B[,2:17],
threshold.df = t.n.df)
exon.cls.df <- mergeCls(compartmentCls = exon.comp.cls,
neighborhoodCls = exon.neigh.cls)
#same order
exon.cls.df <- exon.cls.df[rownames(exon.A),]
# we will add gene symbols as well as peptide count
# (PSM count is also accepted) in case for comparing with
# gene-centric classifications
exon.cls.df$GeneSymbol <- exon.A$Gene_Symbol
exon.cls.df$PeptideCount <- hcc827exon$PeptideCounthead(exon.cls.df)## Protein NeighborhoodCls CompartmentCls Secretory
## ENSE00000331191 ENSE00000331191 Cytosol Unclassified 0.014832469
## ENSE00000331854 ENSE00000331854 Cytosol Unclassified 0.009278669
## ENSE00000331855 ENSE00000331855 Cytosol Unclassified 0.020407456
## ENSE00000331859 ENSE00000331859 Cytosol Unclassified 0.017173531
## ENSE00000331990 ENSE00000331990 Cytosol Unclassified 0.012312932
## ENSE00000332881 ENSE00000332881 Secretory Unclassified 0.916146239
## Nuclear Cytosol Mitochondria S1 S2
## ENSE00000331191 0.02279522 0.956085749 0.006286562 0.004519423 0.004117834
## ENSE00000331854 0.01318771 0.973521876 0.004011749 0.002772796 0.002635333
## ENSE00000331855 0.02129554 0.952790918 0.005506091 0.006079970 0.004366019
## ENSE00000331859 0.01947592 0.957615992 0.005734562 0.005211156 0.004031744
## ENSE00000331990 0.01937522 0.962862663 0.005449185 0.003536029 0.003621272
## ENSE00000332881 0.01224056 0.007886533 0.063726674 0.003485418 0.048403049
## S3 S4 N1 N2 N3
## ENSE00000331191 0.003371211 0.002824002 0.004648321 0.003236997 0.003644227
## ENSE00000331854 0.002135101 0.001735439 0.004252239 0.002268855 0.002583707
## ENSE00000331855 0.005846533 0.004114934 0.006833201 0.002990136 0.003239354
## ENSE00000331859 0.004496494 0.003434136 0.005684534 0.003019969 0.003403090
## ENSE00000331990 0.002855838 0.002299793 0.004253858 0.002914563 0.003272068
## ENSE00000332881 0.211302289 0.652955482 0.002453825 0.005093238 0.002598370
## N4 C1 C2 C3 C4
## ENSE00000331191 0.011265675 0.047842854 0.024531307 0.266857757 0.060419555
## ENSE00000331854 0.004082905 0.525834771 0.396835016 0.042711381 0.002802567
## ENSE00000331855 0.008232845 0.374392624 0.513289731 0.054718728 0.003373434
## ENSE00000331859 0.007368322 0.436126244 0.410295371 0.098762837 0.004447041
## ENSE00000331990 0.008934731 0.479872458 0.212323124 0.188181776 0.011141407
## ENSE00000332881 0.002095123 0.001659707 0.001598117 0.001446198 0.001439449
## C5 M1 M2 GeneSymbol PeptideCount
## ENSE00000331191 0.556434275 0.002860280 0.003426282 CDC45 1
## ENSE00000331854 0.005338141 0.002005498 0.002006251 COPB1 5
## ENSE00000331855 0.007016401 0.002588313 0.002917777 COPB1 1
## ENSE00000331859 0.007984499 0.002584865 0.003149697 COPB1 1
## ENSE00000331990 0.071343897 0.002559130 0.002890055 LPIN1 1
## ENSE00000332881 0.001743062 0.039156448 0.024570225 PTPRA 36.2 Comparison between gene and exon centric classification
The insteresting part of the exon classification is to detect deviated
classification between gene centric classification. However, we enrich
more noise than gene centric classification due to analtyical dept of the exon
centric data. Therefore, we, additioanlly, internally calculate correlation
between gene-centric data hcc827Ctrl and exon-centric data hcc827exon,
and report
number of peptides (PSM count works, too) that match to corresponding exon
as an indication of the confidence of the results.
comp.df <- compareCls(geneCls = cls.df, exonCls = exon.cls.df)head(comp.df)## Exon_id GeneSymbol Gene_Neighborhood Exon_Neighborhood
## 1 ENSE00000400044 ABCF2 Nuclear Nuclear
## 2 ENSE00000400045 ABCF2 Nuclear Cytosol
## 3 ENSE00000400052 ABCF2 Nuclear Cytosol
## 4 ENSE00000400054 ABCF2 Nuclear Nuclear
## 5 ENSE00000653853 ACIN1 Nuclear Nuclear
## 6 ENSE00000655866 ACO2 Mitochondria Mitochondria
## Gene_Compartment Exon_Compartment PeptideCount Pearson.Corr
## 1 N4 N4 2 0.9632687
## 2 N4 C2 6 0.8533751
## 3 N4 C2 1 0.2251107
## 4 N4 N4 2 0.9894984
## 5 N2 N2 21 0.9903756
## 6 M1 M1 2 0.9323598You can easily vizualize/filter the results using correlations and number of peptides.
7 References
Appendix
- Orre., et al. “SubCellBarCode: Proteome-wide Mapping of Protein Localization and Relocalization.” Molecular Cell (2019): 73(1):166-182.e7.
A Session Information
sessionInfo()## R Under development (unstable) (2024-10-21 r87258)
## Platform: x86_64-pc-linux-gnu
## Running under: Ubuntu 24.04.1 LTS
##
## Matrix products: default
## BLAS: /home/biocbuild/bbs-3.21-bioc/R/lib/libRblas.so
## LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.12.0
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## [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
##
## time zone: America/New_York
## tzcode source: system (glibc)
##
## attached base packages:
## [1] stats graphics grDevices utils datasets methods base
##
## other attached packages:
## [1] SubCellBarCode_1.23.0 BiocStyle_2.35.0
##
## loaded via a namespace (and not attached):
## [1] DBI_1.2.3 pROC_1.18.5 gridExtra_2.3
## [4] rlang_1.1.4 magrittr_2.0.3 e1071_1.7-16
## [7] compiler_4.5.0 RSQLite_2.3.7 png_0.1-8
## [10] vctrs_0.6.5 reshape2_1.4.4 stringr_1.5.1
## [13] pkgconfig_2.0.3 crayon_1.5.3 fastmap_1.2.0
## [16] magick_2.8.5 XVector_0.47.0 labeling_0.4.3
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## [22] UCSC.utils_1.3.0 tinytex_0.53 purrr_1.0.2
## [25] bit_4.5.0 xfun_0.48 zlibbioc_1.53.0
## [28] cachem_1.1.0 GenomeInfoDb_1.43.0 jsonlite_1.8.9
## [31] recipes_1.1.0 blob_1.2.4 highr_0.11
## [34] parallel_4.5.0 R6_2.5.1 bslib_0.8.0
## [37] stringi_1.8.4 parallelly_1.38.0 rpart_4.1.23
## [40] lubridate_1.9.3 jquerylib_0.1.4 Rcpp_1.0.13
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## [46] future.apply_1.11.3 IRanges_2.41.0 igraph_2.1.1
## [49] Matrix_1.7-1 splines_4.5.0 nnet_7.3-19
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