Contents

0.1 Introduction

MSstatsResponse provides statistical tools for detecting drug-protein interactions and estimating IC50 values from dose-response mass spectrometry-based proteomics experiments. The package implements isotonic regression models to detect monotonic dose-response relationships and provides robust statistical inference through F-tests and bootstrap confidence intervals.

MSstatsResponse is designed to work with summarized data from MSstats and MSstatsTMT workflows, but can also process other dose-response proteomics datasets that include protein-level quantifications across drug treatments.

0.1.1 Statistical framework

The package uses isotonic regression, a non-parametric method that fits monotonic functions without assuming a specific functional form. This approach is particularly suitable for dose-response data where monotonicity (increasing or decreasing trend) is expected.

0.1.2 Analysis workflow

MSstatsResponse implements the following statistical analysis pipeline:

  1. Data preparation: MSstatsPrepareDoseResponseFit() - Convert input data to standardized format
  2. Interaction detection: doseResponseFit() - Detect drug-protein interactions using isotonic regression and F-tests
  3. IC50 estimation: predictIC50() - Estimate IC50 values with bootstrap confidence intervals
  4. Experimental design planning: futureExperimentSimulation() and run_tpr_simulation() - Simulate dose-response experiments to determine optimal study designs

0.2 Installation and setup

0.2.1 Install MSstatsResponse

if (!requireNamespace("BiocManager", quietly = TRUE))
  install.packages("BiocManager")

BiocManager::install("MSstatsResponse")

0.2.2 Load required libraries

library(MSstatsResponse)
library(dplyr)
library(ggplot2)
library(data.table)

# Optional: for upstream data processing
# library(MSstats)
# library(MSstatsTMT)

0.3 Data preprocessing with MSstats

When analyzing a mass spectrometry-based proteomics dataset, data should be preprocessed using the standard MSstats workflow for cleaning, normalization, and protein summarization prior to analysis with MSstatsResponse.

When using MSstats, data can be converted with one of the available converter functions (e.g., SpectronauttoMSstatsFormat()) and subsequently processed with dataProcess().

For MSstatsTMT, converter functions (e.g., MaxQtoMSstatsTMTFormat()) may be used, followed by proteinSummarization() for data processing.

Please refer to the MSstats and MSstatsTMT Bioconductor vignettes for a comprehensive list of supported converters and detailed preprocessing recommendations.

0.3.1 Example MSstats workflow

# Read raw data (example with Spectronaut output)
raw_data <- read_tsv("path/to/spectronaut_report.tsv")

# Convert to MSstats format
msstats_data <- SpectronauttoMSstatsFormat(raw_data)

# Process data: normalization and protein summarization
processed_data <- dataProcess(
  msstats_data,
  normalization = "equalizeMedians",
  summaryMethod = "TMP",
  MBimpute = TRUE,
  maxQuantileforCensored = 0.999
)

# Extract protein-level data for dose-response analysis
protein_level_data <- processed_data$ProteinLevelData

0.3.2 Loading example data

For this vignette, we use pre-processed data prepared following the steps described above. For more information on preprocessing mass spectrometry-based proteomics experiments, see the vignettes for MSstats and/or MSstatsTMT.

data("DIA_MSstats_Normalized", package = "MSstatsResponse")
dia_normalized <- DIA_MSstats_Normalized

str(dia_normalized$ProteinLevelData[1:5, ])
#> 'data.frame':    5 obs. of  11 variables:
#>  $ RUN                   : Factor w/ 27 levels "1","2","3","4",..: 1 2 3 4 5
#>  $ Protein               : Factor w/ 3 levels "PROTEIN_A","PROTEIN_B",..: 1 1 1 1 1
#>  $ LogIntensities        : num  5.05 5.65 5.15 5.02 5.32
#>  $ originalRUN           : Factor w/ 27 levels "20240221_3uL_neu-01_dmso-1_dia_2x10_0p65-1p45_85min_Slot1-1_1_2571",..: 1 2 3 4 5
#>  $ GROUP                 : Factor w/ 9 levels "Drug1_001nM",..: 9 9 9 1 1
#>  $ SUBJECT               : num  1 2 3 1 2
#>  $ TotalGroupMeasurements: int  396 396 396 396 396
#>  $ NumMeasuredFeature    : int  132 132 132 132 132
#>  $ MissingPercentage     : num  0 0 0 0 0
#>  $ more50missing         : logi  FALSE FALSE FALSE FALSE FALSE
#>  $ NumImputedFeature     : int  0 0 0 0 0

0.4 Data preparation for dose-response analysis

0.4.1 Converting GROUP labels to numeric doses

The convertGroupToNumericDose() function parses MSstats-style GROUP labels to extract drug names and numeric dose values.

Format expectations:

  • Control samples: "DMSO" (assigned dose = 0)
  • Treatment samples: "DrugName_ValueUnit" (e.g., "Drug1_003uM", "Drug2_300nM")
dose_info <- convertGroupToNumericDose(dia_normalized$ProteinLevelData$GROUP)

dia_normalized$ProteinLevelData <- dia_normalized$ProteinLevelData %>%
  mutate(
    dose_nM = dose_info$dose_nM,
    drug = dose_info$drug
  )

dia_normalized$ProteinLevelData %>%
  select(Protein, GROUP, drug, dose_nM) %>%
  head(10)
#>      Protein       GROUP  drug dose_nM
#> 1  PROTEIN_A        DMSO  DMSO       0
#> 2  PROTEIN_A        DMSO  DMSO       0
#> 3  PROTEIN_A        DMSO  DMSO       0
#> 4  PROTEIN_A Drug1_001nM Drug1       1
#> 5  PROTEIN_A Drug1_001nM Drug1       1
#> 6  PROTEIN_A Drug1_001nM Drug1       1
#> 7  PROTEIN_A Drug1_001uM Drug1    1000
#> 8  PROTEIN_A Drug1_001uM Drug1    1000
#> 9  PROTEIN_A Drug1_001uM Drug1    1000
#> 10 PROTEIN_A Drug1_003nM Drug1       3

0.4.2 Formatting data with MSstatsPrepareDoseResponseFit()

This function standardizes the data structure for downstream analysis. The dose_column corresponds to the x-axis values (e.g., dose, concentration, time, or temperature), the protein_column should contain unique identifiers (e.g., protein, gene, or peptide sequence), and the log_abundance_column represents the response values on the y-axis (e.g., log intensity, expression, or growth).

dia_prepared <- MSstatsPrepareDoseResponseFit(
  data = dia_normalized$ProteinLevelData,
  dose_column = "dose_nM",
  drug_column = "drug",
  protein_column = "Protein",
  log_abundance_column = "LogIntensities",
  transform_nM_to_M = TRUE
)

str(dia_prepared)
#> 'data.frame':    81 obs. of  5 variables:
#>  $ protein : Factor w/ 3 levels "PROTEIN_A","PROTEIN_B",..: 1 1 1 1 1 1 1 1 1 1 ...
#>  $ drug    : chr  "DMSO" "DMSO" "DMSO" "Drug1" ...
#>  $ dose    : num  0e+00 0e+00 0e+00 1e-09 1e-09 1e-09 1e-06 1e-06 1e-06 3e-09 ...
#>  $ response: num  5.05 5.65 5.15 5.02 5.32 ...
#>  $ dose_nM : num  0 0 0 1 1 1 1000 1000 1000 3 ...

dia_prepared %>%
  filter(protein %in% c("PROTEIN_A", "PROTEIN_B")) %>%
  arrange(protein, drug, dose) %>%
  head(20)
#>      protein  drug  dose response dose_nM
#> 1  PROTEIN_A  DMSO 0e+00 5.050662       0
#> 2  PROTEIN_A  DMSO 0e+00 5.647209       0
#> 3  PROTEIN_A  DMSO 0e+00 5.148809       0
#> 4  PROTEIN_A Drug1 1e-09 5.019425       1
#> 5  PROTEIN_A Drug1 1e-09 5.318420       1
#> 6  PROTEIN_A Drug1 1e-09 5.141087       1
#> 7  PROTEIN_A Drug1 3e-09 5.218344       3
#> 8  PROTEIN_A Drug1 3e-09 5.199884       3
#> 9  PROTEIN_A Drug1 3e-09 5.320023       3
#> 10 PROTEIN_A Drug1 1e-08 4.848356      10
#> 11 PROTEIN_A Drug1 1e-08 5.205914      10
#> 12 PROTEIN_A Drug1 1e-08 4.743209      10
#> 13 PROTEIN_A Drug1 3e-08 4.663491      30
#> 14 PROTEIN_A Drug1 3e-08 4.486120      30
#> 15 PROTEIN_A Drug1 3e-08 4.505798      30
#> 16 PROTEIN_A Drug1 1e-07 3.936098     100
#> 17 PROTEIN_A Drug1 1e-07 4.231085     100
#> 18 PROTEIN_A Drug1 1e-07 4.327733     100
#> 19 PROTEIN_A Drug1 3e-07 3.590255     300
#> 20 PROTEIN_A Drug1 3e-07 3.212578     300

0.5 Drug-protein interaction detection

0.5.1 Isotonic regression

Isotonic regression fits a monotonic function to the data without assuming a parametric form. For inhibitor data, we typically expect a non-increasing response: drug binding reduces protein abundance monotonically with dose.

0.5.2 Running doseResponseFit()

The doseResponseFit() function:

  1. Fits isotonic regression for each protein-drug pair
  2. Performs an F-test comparing the fitted model to a flat (null) model
  3. Adjusts p-values for multiple testing using the Benjamini-Hochberg procedure
response_results <- doseResponseFit(
  data = dia_prepared,
  increasing = FALSE,
  transform_dose = TRUE,
  ratio_response = FALSE
)
#> 
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response_results %>%
  select(Protein, drug, F_statistic, log2FC, adj.pvalue) %>%
  arrange(adj.pvalue) %>%
  head(10)
#>     Protein  drug F_statistic    log2FC   adj.pvalue
#> 1 PROTEIN_B Drug1  255.431210 -5.201664 0.000000e+00
#> 2 PROTEIN_A Drug1   76.470195 -2.515487 3.885392e-12
#> 3 PROTEIN_C Drug1    1.180529 -0.105177 3.627180e-01

0.5.3 Interpreting results

  • log2FC: Log2 fold change between the highest and lowest doses
  • F_statistic: Ratio of between-dose to within-dose variance
  • P_value: Raw p-value from F-test
  • adj.pvalue: FDR-adjusted p-value (Benjamini-Hochberg)

Proteins with adj.pvalue < 0.05 show significant dose-dependent responses.

n_total <- nrow(response_results)
n_significant <- sum(response_results$adj.pvalue < 0.05)

cat("Total protein-drug pairs tested:", n_total, "\n")
#> Total protein-drug pairs tested: 3
cat("Significant interactions (FDR < 0.05):", n_significant, "\n")
#> Significant interactions (FDR < 0.05): 2
cat("Percentage significant:", round(100 * n_significant / n_total, 1), "%\n")
#> Percentage significant: 66.7 %

top_hits <- response_results %>%
  filter(adj.pvalue < 0.05) %>%
  arrange(adj.pvalue) %>%
  head(5)

print(top_hits)
#>     Protein  drug  direction  SSE_Full  SSE_Null F_statistic      P_value
#> 1 PROTEIN_B Drug1 decreasing 1.0502856 120.28394    255.4312 0.000000e+00
#> 2 PROTEIN_A Drug1 decreasing 0.7482337  26.17827     76.4702 2.590261e-12
#>      log2FC   adj.pvalue
#> 1 -5.201664 0.000000e+00
#> 2 -2.515487 3.885392e-12

0.6 IC50 estimation

0.6.1 Understanding IC50 calculation

IC50 represents the dose at which the response is reduced to 50% of the control (DMSO) level. MSstatsResponse estimates IC50 by:

  1. Fitting isotonic regression on the ratio scale (response relative to DMSO)
  2. Finding the dose where the fitted response equals 0.5
  3. Computing confidence intervals via bootstrap resampling

0.6.2 Running predictIC50()

ic50_predictions <- predictIC50(
  data = dia_prepared,
  ratio_response = TRUE,
  transform_dose = TRUE,
  increasing = FALSE,
  bootstrap = TRUE,
  n_samples = 1000,
  alpha = 0.10
)

ic50_predictions %>%
  arrange(IC50) %>%
  head(10)
#>      Protein   drug    IC50 IC50_lower_bound IC50_upper_bound  direction
#>       <fctr> <char>   <num>            <num>            <num>     <char>
#> 1: PROTEIN_A  Drug1 7.47197         7.888761         7.198295 decreasing
#> 2: PROTEIN_B  Drug1 8.19419         8.513953         7.950345 decreasing
#> 3: PROTEIN_C  Drug1      NA               NA               NA decreasing

0.6.3 Parallel processing for large datasets

For datasets with many proteins, use parallel processing via BiocParallel:

library(BiocParallel)
if (.Platform$OS.type == "windows") {
  register(SnowParam(workers = 4, type = "SOCK"))
} else {
  register(MulticoreParam(workers = 4))
}

ic50_parallel <- predictIC50(
  data = dia_prepared,
  ratio_response = TRUE,
  transform_dose = TRUE,
  bootstrap = TRUE,
  n_samples = 1000,
  BPPARAM = bpparam()
)

0.7 Visualization

0.7.1 Individual protein dose-response curves

The visualizeResponseProtein() function creates dose-response plots:

visualizeResponseProtein(
  data = dia_prepared,
  protein_name = "PROTEIN_A",
  drug_name = "Drug1",
  ratio_response = TRUE,
  show_ic50 = TRUE,
  add_ci = TRUE,
  transform_dose = TRUE,
  n_samples = 1000
)

visualizeResponseProtein(
  data = dia_prepared,
  protein_name = "PROTEIN_B",
  drug_name = "Drug1",
  ratio_response = TRUE,
  show_ic50 = TRUE,
  add_ci = TRUE
)

0.7.2 Comparing log2 vs ratio scale

p1 <- visualizeResponseProtein(
  data = dia_prepared,
  protein_name = "PROTEIN_A",
  drug_name = "Drug1",
  ratio_response = FALSE,
  show_ic50 = TRUE,
  add_ci = FALSE
)

p2 <- visualizeResponseProtein(
  data = dia_prepared,
  protein_name = "PROTEIN_A",
  drug_name = "Drug1",
  ratio_response = TRUE,
  show_ic50 = TRUE,
  add_ci = FALSE
)

gridExtra::grid.arrange(p1, p2, ncol = 2)

0.7.3 Predicting at other response levels

The target_response argument allows prediction at any response level (default 0.50). This example predicts IC25 with confidence intervals:

visualizeResponseProtein(
  data = dia_prepared,
  protein_name = "PROTEIN_B",
  drug_name = "Drug1",
  ratio_response = TRUE,
  show_ic50 = TRUE,
  add_ci = TRUE,
  transform_dose = TRUE,
  target_response = 0.25
)

0.8 Experimental design planning

0.8.1 Simulating future experiments

futureExperimentSimulation() evaluates detection performance under a user-defined experimental design by simulating a full experiment and measuring true-positive and false-positive rates. Proteins from your own data can be used as templates for more realistic simulations.

# pull unique doses 
dose_levels = as.numeric(unique(dia_prepared$dose))

# run simulation
custom_simulation <- futureExperimentSimulation(
  N_proteins = 300,
  N_rep = 2,
  N_Control_Rep = 2,
  Concentrations = dose_levels,
  data = dia_prepared,
  strong_proteins = "PROTEIN_B",
  weak_proteins = "PROTEIN_A",
  no_interaction_proteins = "PROTEIN_C",
  drug_name = "Drug1",
  IC50_Prediction = FALSE
)
#> 
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print(custom_simulation$Hit_Rates_Plot)

print(custom_simulation$Template_Used)
#> $strong_interaction
#>    dose Intensity LogIntensities      ratio
#> 1 0e+00 42.256113      5.4010882 1.00000000
#> 2 1e-09 38.863432      5.2803414 0.91971147
#> 3 1e-06  1.266790      0.3411772 0.02997885
#> 4 3e-09 26.873610      4.7481182 0.63596977
#> 5 3e-06  1.177035      0.2351572 0.02785479
#> 6 1e-08 22.303763      4.4792152 0.52782334
#> 7 3e-08 17.327563      4.1149968 0.41006050
#> 8 1e-07 10.777063      3.4298922 0.25504152
#> 9 3e-07  1.186329      0.2465041 0.02807473
#> 
#> $weak_interaction
#>    dose Intensity LogIntensities     ratio
#> 1 0e+00 38.914246       5.282227 1.0000000
#> 2 1e-09 35.744368       5.159644 0.9185420
#> 3 1e-06  7.395559       2.886659 0.1900476
#> 4 3e-09 37.951462       5.246084 0.9752588
#> 5 3e-06  6.878926       2.782183 0.1767714
#> 6 1e-08 30.537135       4.932493 0.7847289
#> 7 3e-08 23.454666       4.551803 0.6027270
#> 8 1e-07 17.938304       4.164972 0.4609701
#> 9 3e-07  9.453305       3.240819 0.2429266
#> 
#> $no_interaction
#>    dose Intensity LogIntensities     ratio
#> 1 0e+00  59.16019       5.886555 1.0000000
#> 2 1e-09  60.11579       5.909672 1.0161526
#> 3 1e-06  56.94126       5.831402 0.9624927
#> 4 3e-09  56.23066       5.813285 0.9504813
#> 5 3e-06  55.61656       5.797443 0.9401010
#> 6 1e-08  57.99096       5.857756 0.9802362
#> 7 3e-08  57.62351       5.848586 0.9740250
#> 8 1e-07  57.80438       5.853107 0.9770823
#> 9 3e-07  54.28170       5.762394 0.9175376

0.8.2 Sweeping experimental design parameters

run_tpr_simulation() sweeps over combinations of replicate counts and number of concentration levels, measuring the true positive rate (TPR) for each design configuration. Control (0) and the highest dose are always included; intermediate doses are selected by greedily choosing the concentration farthest from the log-median of the already-selected set.

Use plot_tpr_power_curve() to visualize the resulting power curves.

power_results <- run_tpr_simulation(
  rep_range = c(2,4),
  concentrations = dose_levels,
  dose_range = c(2, 9),
  data = dia_prepared,
  protein = "PROTEIN_A",
  n_proteins = 300
)
#> 
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plot_tpr_power_curve(power_results)

0.9 Session information

sessionInfo()
#> R version 4.6.0 RC (2026-04-17 r89917)
#> Platform: x86_64-pc-linux-gnu
#> Running under: Ubuntu 24.04.4 LTS
#> 
#> Matrix products: default
#> BLAS:   /home/biocbuild/bbs-3.23-bioc/R/lib/libRblas.so 
#> LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.12.0  LAPACK version 3.12.0
#> 
#> locale:
#>  [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
#>  [3] LC_TIME=en_GB              LC_COLLATE=C              
#>  [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
#>  [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
#>  [9] LC_ADDRESS=C               LC_TELEPHONE=C            
#> [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] data.table_1.18.2.1   ggplot2_4.0.3         dplyr_1.2.1          
#> [4] MSstatsResponse_1.1.2 BiocStyle_2.39.0     
#> 
#> loaded via a namespace (and not attached):
#>  [1] plotly_4.12.0       sass_0.4.10         generics_0.1.4     
#>  [4] tidyr_1.3.2         stringi_1.8.7       digest_0.6.39      
#>  [7] magrittr_2.0.5      evaluate_1.0.5      grid_4.6.0         
#> [10] RColorBrewer_1.1-3  bookdown_0.46       fastmap_1.2.0      
#> [13] jsonlite_2.0.0      tinytex_0.59        gridExtra_2.3      
#> [16] BiocManager_1.30.27 httr_1.4.8          purrr_1.2.2        
#> [19] crosstalk_1.2.2     viridisLite_0.4.3   scales_1.4.0       
#> [22] codetools_0.2-20    lazyeval_0.2.3      jquerylib_0.1.4    
#> [25] cli_3.6.6           rlang_1.2.0         withr_3.0.2        
#> [28] cachem_1.1.0        yaml_2.3.12         otel_0.2.0         
#> [31] tools_4.6.0         parallel_4.6.0      BiocParallel_1.45.0
#> [34] vctrs_0.7.3         R6_2.6.1            magick_2.9.1       
#> [37] lifecycle_1.0.5     stringr_1.6.0       htmlwidgets_1.6.4  
#> [40] pkgconfig_2.0.3     pillar_1.11.1       bslib_0.10.0       
#> [43] gtable_0.3.6        Rcpp_1.1.1-1        glue_1.8.1         
#> [46] xfun_0.57           tibble_3.3.1        tidyselect_1.2.1   
#> [49] knitr_1.51          dichromat_2.0-0.1   farver_2.1.2       
#> [52] htmltools_0.5.9     labeling_0.4.3      rmarkdown_2.31     
#> [55] compiler_4.6.0      S7_0.2.2

0.10 References

  1. Szvetecz, S., et al. (2026). MSstatsResponse: Semi-parametric statistical model enhances drug-protein interaction detection and IC50 estimation in chemoproteomics experiments. Manuscript in preparation.

  2. Choi, M., et al. (2014). MSstats: an R package for statistical analysis of quantitative mass spectrometry-based proteomic experiments. Bioinformatics, 30(17), 2524-2526.