Introduction to TPP2D for 2D-TPP analysis
28 October, 2020
Package
TPP2D 1.6.0
Contents
1 Abstract
Thermal proteome profiling (TPP) (Mateus et al., 2020; Savitski et al., 2014) is an unbiased mass spectrometry-based method to assess protein-ligand interactions. It works by employing the cellular thermal shift assay (CETSA) (Molina et al., 2013) on a proteome-wide scale which monitors the profiles of proteins in cells over a temperature gradient and aims to detect shifts induced by ligand-protein interactions. 2D-TPP represents a refined version of the assay (Becher et al., 2016) which uses a concentration gradient of the ligand of interest over a temperature gradient. This package aims to analyze data retrieved from 2D-TPP experiments by a functional analysis approach.
2 General information
This package implements a method to detect ligand-protein interactions from
datasets obtained with the 2D-TPP assay. Please note that methods for analyzing
convential TPP datasets (e.g. single dose, melting curve approach) can be found
at: TPP and
NPARC .
This vignette is not aiming to give an in-depth introduction to thermal proteome profiling, please refer to other sources for this purpose:
Note: if you use TPP2D in published research, please cite:
Kurzawa, N.*, Becher, I.* et al. (2020) Computational analysis of ligand dose range thermal proteome profiles. bioRxiv, 10.1101/2020.05.08.083709
3 Installation
- Download the package from Bioconductor.
if (!requireNamespace("BiocManager", quietly = TRUE))
install.packages("BiocManager")
BiocManager::install("TPP2D")Or install the development version of the package from Github.
BiocManager::install(“nkurzaw/TPP2D”)- Load the package into R session.
library(TPP2D)4 Introduction
The 2D-TPP assay is usually set up in a way that for each temperature different ligand concentrations (including a vehicle condition) are used and two adjacent temperatures each are multiplexed in a single mass spectrometry (MS) run. Typically up to 10 or 12 temperatures are used in total that add up to 5 or 6 MS runs respectively (Figure 1).
Figure 1: Experimental 2D-TPP workflow
This package aims to provide a tool for finding ligand-protein interactions, i.e. proteins affected in their thermal stability by the treatment used in the experiment) at a given false disscovery rate (FDR). Please note that a change in thermal stability of a protein is not a guarantee for it interacting with the molecule used as treatment. However, we try to give the user additional information by specifying whether an observed effect is likely due to stabilization or a change in expression or solubility of a given protein to make the interpretation of detected hits as easy as possible.
5 Step-by-step workflow
library(dplyr)
library(TPP2D)After having loaded dplyr and the TPP2D package itself we start by loading
an example dataset which is supplied with the package. Therefore, we use
the import2dDataset function.
For this puporse we need to supply a config table that essentially describes
which experimental conditions the different TMT labels used correspond to and
supplies paths to the raw data files (note: since this example dataset is
included in the package it does not contain a “Path” column, this is however
mandatory if the data should be read in from external raw files).
data("config_tab")
data("raw_dat_list")
config_tab## Compound Experiment Temperature 126 127L 127H 128L 128H 129L 129H 130L 130H
## 1 Compound1 exp1 37.0 0 0.5 2 10 40 - - - -
## 2 Compound1 exp1 37.8 - - - - - 0 0.5 2 10
## 3 Compound1 exp2 40.4 0 0.5 2 10 40 - - - -
## 4 Compound1 exp2 44.0 - - - - - 0 0.5 2 10
## 5 Compound1 exp3 46.9 0 0.5 2 10 40 - - - -
## 6 Compound1 exp3 49.8 - - - - - 0 0.5 2 10
## 7 Compound1 exp4 52.9 0 0.5 2 10 40 - - - -
## 8 Compound1 exp4 55.5 - - - - - 0 0.5 2 10
## 9 Compound1 exp5 58.6 0 0.5 2 10 40 - - - -
## 10 Compound1 exp5 62.0 - - - - - 0 0.5 2 10
## 11 Compound1 exp6 65.4 0 0.5 2 10 40 - - - -
## 12 Compound1 exp6 66.3 - - - - - 0 0.5 2 10
## 131L RefCol
## 1 - 126
## 2 40 129L
## 3 - 126
## 4 40 129L
## 5 - 126
## 6 40 129L
## 7 - 126
## 8 40 129L
## 9 - 126
## 10 40 129L
## 11 - 126
## 12 40 129LWe then call the import function (note: we here supply a list of data frames for the “data” argument, replacing the raw data files that would be normally specified in the above mentioned column of the config table. If this is supplied the argument “data” can simply be ignored):
import_df <- import2dDataset(
configTable = config_tab,
data = raw_dat_list,
idVar = "protein_id",
intensityStr = "signal_sum_",
fcStr = "rel_fc_",
nonZeroCols = "qusm",
geneNameVar = "gene_name",
addCol = NULL,
qualColName = "qupm",
naStrs = c("NA", "n/d", "NaN"),
concFactor = 1e6,
medianNormalizeFC = TRUE,
filterContaminants = TRUE)## The following valid label columns were detected:
## 126, 127L, 127H, 128L, 128H, 129L, 129H, 130L, 130H, 131L.## Importing 2D-TPP dataset: exp1## Removing duplicate identifiers using quality column 'qupm'...## 20 out of 20 rows kept for further analysis.## Importing 2D-TPP dataset: exp1## Removing duplicate identifiers using quality column 'qupm'...## 20 out of 20 rows kept for further analysis.## Importing 2D-TPP dataset: exp2## Removing duplicate identifiers using quality column 'qupm'...## 20 out of 20 rows kept for further analysis.## Importing 2D-TPP dataset: exp2## Removing duplicate identifiers using quality column 'qupm'...## 20 out of 20 rows kept for further analysis.## Importing 2D-TPP dataset: exp3## Removing duplicate identifiers using quality column 'qupm'...## 15 out of 15 rows kept for further analysis.## Importing 2D-TPP dataset: exp3## Removing duplicate identifiers using quality column 'qupm'...## 15 out of 15 rows kept for further analysis.## Importing 2D-TPP dataset: exp4## Removing duplicate identifiers using quality column 'qupm'...## 14 out of 14 rows kept for further analysis.## Importing 2D-TPP dataset: exp4## Removing duplicate identifiers using quality column 'qupm'...## 14 out of 14 rows kept for further analysis.## Importing 2D-TPP dataset: exp5## Removing duplicate identifiers using quality column 'qupm'...## 9 out of 9 rows kept for further analysis.## Importing 2D-TPP dataset: exp5## Removing duplicate identifiers using quality column 'qupm'...## 9 out of 9 rows kept for further analysis.## Importing 2D-TPP dataset: exp6## Removing duplicate identifiers using quality column 'qupm'...## 4 out of 4 rows kept for further analysis.## Importing 2D-TPP dataset: exp6## Removing duplicate identifiers using quality column 'qupm'...## 4 out of 4 rows kept for further analysis.## Ratios were correctly computed!## Median normalizing fold changes...recomp_sig_df <- recomputeSignalFromRatios(import_df)
# resolve ambiguous protein names
preproc_df <- resolveAmbiguousProteinNames(recomp_sig_df)
# alternatively one could choose to run
# preproc_df <- resolveAmbiguousProteinNames(
# recomp_sig_df, includeIsoforms = TRUE)Please refer to the help page of the function to retrieve in-depth description
of the different arguments. Essentially the function needs to know the names
or prefixes of the columns in the raw data files, that contain different
informations like protein id or the raw or relative signal intensities
measured for the different TMT labels.
The imported synthetic dataset consists of 17 simulated protein 2D thermal
profiles (protein1-17) and 3 spiked-in true positives (tp1-3). It represents
a data frame with the columns:
| column | description | required |
|---|---|---|
| representative | protein identifier | Yes |
| qupm | number of unique quantified peptides | No |
| qusm | number of unique spectra | No |
| clustername | gene name | Yes |
| temperature | temperature incubated at | Yes |
| experiment | experiment identifier | No |
| label | TMT label | No |
| RefCol | RefCol | No |
| conc | treatment concentration | No |
| raw_value | raw reporter ion intensity sum | No |
| raw_rel_value | raw relative fold change compared to vehicle condition at the same temperature | No |
| log_conc | log10 treatment concentration | Yes |
| rel_value | median normalized fold change | No |
| value | recomputed reporter ion intensity | No |
| log2_value | recomputed log2 reporter ion intensity | Yes |
Here the column “required” indicates which of these columns is neccessary for usage of the downstream functions.
We then begin our actual data analysis by fitting two nested models to each protein profile: A null model that is expected when a protein profile remains unaffected by a given treatment and an alternative model which is a constrained sigmoidal dose-response model across all temperatures.
model_params_df <- getModelParamsDf(
df = preproc_df)The goodness of fit of both models for each protein is then compared and a \(F\)-statistic is computed.
fstat_df <- computeFStatFromParams(model_params_df)Then we create a null model using our dataset to be able to estimate the FDR for a given \(F\)-statistic in the next step.
set.seed(12, kind = "L'Ecuyer-CMRG")
null_model <- bootstrapNullAlternativeModel(
df = preproc_df,
params_df = model_params_df,
B = 2)## [1] "Warning: You have specificed B < 20, it is recommended to use at least B = 20 in order to obtain reliable results."
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|======================================================================| 100%Please note that setting \(B = 2\) (corresponsing to \(2\) permutations) is not enough to guarantee faithful FDR estimation, this has simply been set for fast demonstration purposes. We recommend to use at least \(B = 20\) for applications in praxis.
To estimate the FDR for all given \(F\)-statistics and retrieve all significant hits at a set FDR \(\alpha\) we use the following functions:
fdr_tab <- getFDR(
df_out = fstat_df,
df_null = null_model)
hits <- findHits(
fdr_df = fdr_tab,
alpha = 0.1)
hits %>%
dplyr::select(clustername, nObs, F_statistic, FDR)## # A tibble: 5 x 4
## clustername nObs F_statistic FDR
## <chr> <int> <dbl> <dbl>
## 1 tp2 30 53.9 0
## 2 tp1 20 47.7 0
## 3 tp3 50 31.3 0
## 4 protein10 50 2.42 0
## 5 protein15 50 1.91 0We can now plot our obtained result as a (one-sided) volcano plot:
plot2dTppVolcano(fdr_df = fdr_tab, hits_df = hits)
Finally, we can fit and plot proteins that have come up as significant in our analysis by using:
plot2dTppFit(recomp_sig_df, "tp1", model_type = "H0")
or respectively for the H1 model:
plot2dTppFit(recomp_sig_df, "tp1", model_type = "H1")
An alternative option to visualize thermal profiles of proteins of interest is to use plot2dTppFcHeatmap:
plot2dTppFcHeatmap(recomp_sig_df, "tp1",
drug_name = "drug X")
plot2dTppFcHeatmap(recomp_sig_df, "tp3",
drug_name = "drug X")
6 Acknowledgements
We thank the following people for valuable feedback and help with the TPP2D package:
- André Mateus
- Constantin Ahlmann-Eltze
- Alexander Helmboldt
- Stefan Gade
- Dorothee Childs
- Nikos Ignatiadis
- Britta Velten
sessionInfo()## R version 4.0.3 (2020-10-10)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 18.04.5 LTS
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## BLAS: /home/biocbuild/bbs-3.12-bioc/R/lib/libRblas.so
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## Random number generation:
## RNG: L'Ecuyer-CMRG
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## Sample: Rejection
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## attached base packages:
## [1] stats graphics grDevices utils datasets methods base
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## other attached packages:
## [1] TPP2D_1.6.0 dplyr_1.0.2 BiocStyle_2.18.0
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## loaded via a namespace (and not attached):
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## [55] doParallel_1.0.16 crayon_1.3.4References
Becher, I., Werner, T., Doce, C., Zaal, E.A., Tögel, I., Khan, C.A., Rueger, A., Muelbaier, M., Salzer, E., Berkers, C.R., et al. (2016). Thermal profiling reveals phenylalanine hydroxylase as an off-target of panobinostat. Nature Chemical Biology 12, 908–910.
Mateus, A., Kurzawa, N., Becher, I., Sridharan, S., Helm, D., Stein, F., Typas, A., and Savitski, M.M. (2020). Thermal proteome profiling for interrogating protein interactions. Molecular Systems Biology 16, e9232.
Molina, D.M., Jafari, R., Ignatushchenko, M., Seki, T., Larsson, E.A., Dan, C., Sreekumar, L., Cao, Y., and Nordlund, P. (2013). Monitoring Drug Target Engagement in Cells and Tissues Using the Cellular Thermal Shift Assay. Science 341, 84–88.
Savitski, M.M., Reinhard, F.B.M., Franken, H., Werner, T., Savitski, M.F., Eberhard, D., Martinez Molina, D., Jafari, R., Dovega, R.B., Klaeger, S., et al. (2014). Tracking cancer drugs in living cells by thermal profiling of the proteome. Science 346, 1255784.