MSstatsPTM LabelFree Workflow

Devon Kohler (kohler.d@northeastern.edu)

2022-04-26

library(MSstatsPTM)

This Vignette provides an example workflow for how to use the package MSstatsPTM for a labelfree dataset. It also provides examples and an analysis of how adjusting for global protein levels allows for better interpretations of PTM modeling results.

Installation

To install this package, start R (version “4.0”) and enter:

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

BiocManager::install("MSstatsPTM")

1. Workflow

1.1 Raw Data Format

Note: We are actively developing dedicated converters for MSstatsPTM. If you have data from a processing tool that does not have a dedicated converter in MSstatsPTM please add a github issue https://github.com/Vitek-Lab/MSstatsPTM/issues and we will add the converter.

The first step is to load in the raw dataset for both the PTM and Protein datasets. Each dataset can formatted using dedicated converters in MSstatsPTM, such as ProgenesistoMSstatsPTMFormat, or converters from base MSstats such as SkylinetoMSstatsFormat, MaxQtoMSstatsFormat, ProgenesistoMSstatsFormat, ect. If using converters from MSstats note they will need to be run both on the global protein and PTM datasets.

Please note for the PTM dataset, both the protein and modification site (or peptide), must be added into the ProteinName column. This allows for the package to summarize to the peptide level, and avoid the off chance there are matching peptides between proteins. For an example of how this can be done please see the code below.

1.1.1 Raw Data Converter

The output of the converter is a list with two formatted data.tables. One each for the PTM and Protein datasets.

If there is not a dedicated MSstatsPTM converter for a processing tool, base MSstats converters can be used as follows. Please note ProteinName column must be a combination of the Protein Name and sitename.

Both of these conversion methods will output the same results.

1.2 Summarization - dataSummarizationPTM

After loading in the input data, the next step is to use the dataSummarizationPTM function. This provides the summarized dataset needed to model the protein/PTM abundance. The function will summarize the Protein dataset up to the protein level and will summarize the PTM dataset up to the peptide level. There are multiple options for normalization and missing value imputation. These options should be reviewed in the package documentation.


MSstatsPTM.summary <- dataSummarizationPTM(raw.input, verbose = FALSE)
#> 
  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |=                                                                     |   1%
  |                                                                            
  |=                                                                     |   2%
  |                                                                            
  |==                                                                    |   2%
  |                                                                            
  |==                                                                    |   3%
  |                                                                            
  |===                                                                   |   4%
  |                                                                            
  |===                                                                   |   5%
  |                                                                            
  |====                                                                  |   6%
  |                                                                            
  |=====                                                                 |   7%
  |                                                                            
  |======                                                                |   8%
  |                                                                            
  |======                                                                |   9%
  |                                                                            
  |=======                                                               |  10%
  |                                                                            
  |========                                                              |  11%
  |                                                                            
  |========                                                              |  12%
  |                                                                            
  |=========                                                             |  13%
  |                                                                            
  |==========                                                            |  14%
  |                                                                            
  |===========                                                           |  15%
  |                                                                            
  |===========                                                           |  16%
  |                                                                            
  |============                                                          |  17%
  |                                                                            
  |============                                                          |  18%
  |                                                                            
  |=============                                                         |  18%
  |                                                                            
  |=============                                                         |  19%
  |                                                                            
  |==============                                                        |  20%
  |                                                                            
  |===============                                                       |  21%
  |                                                                            
  |===============                                                       |  22%
  |                                                                            
  |================                                                      |  22%
  |                                                                            
  |================                                                      |  23%
  |                                                                            
  |=================                                                     |  24%
  |                                                                            
  |=================                                                     |  25%
  |                                                                            
  |==================                                                    |  26%
  |                                                                            
  |===================                                                   |  27%
  |                                                                            
  |====================                                                  |  28%
  |                                                                            
  |====================                                                  |  29%
  |                                                                            
  |=====================                                                 |  30%
  |                                                                            
  |======================                                                |  31%
  |                                                                            
  |======================                                                |  32%
  |                                                                            
  |=======================                                               |  33%
  |                                                                            
  |========================                                              |  34%
  |                                                                            
  |=========================                                             |  35%
  |                                                                            
  |=========================                                             |  36%
  |                                                                            
  |==========================                                            |  37%
  |                                                                            
  |==========================                                            |  38%
  |                                                                            
  |===========================                                           |  38%
  |                                                                            
  |===========================                                           |  39%
  |                                                                            
  |============================                                          |  40%
  |                                                                            
  |=============================                                         |  41%
  |                                                                            
  |=============================                                         |  42%
  |                                                                            
  |==============================                                        |  42%
  |                                                                            
  |==============================                                        |  43%
  |                                                                            
  |===============================                                       |  44%
  |                                                                            
  |===============================                                       |  45%
  |                                                                            
  |================================                                      |  46%
  |                                                                            
  |=================================                                     |  47%
  |                                                                            
  |==================================                                    |  48%
  |                                                                            
  |==================================                                    |  49%
  |                                                                            
  |===================================                                   |  50%
  |                                                                            
  |====================================                                  |  51%
  |                                                                            
  |====================================                                  |  52%
  |                                                                            
  |=====================================                                 |  53%
  |                                                                            
  |======================================                                |  54%
  |                                                                            
  |=======================================                               |  55%
  |                                                                            
  |=======================================                               |  56%
  |                                                                            
  |========================================                              |  57%
  |                                                                            
  |========================================                              |  58%
  |                                                                            
  |=========================================                             |  58%
  |                                                                            
  |=========================================                             |  59%
  |                                                                            
  |==========================================                            |  60%
  |                                                                            
  |===========================================                           |  61%
  |                                                                            
  |===========================================                           |  62%
  |                                                                            
  |============================================                          |  62%
  |                                                                            
  |============================================                          |  63%
  |                                                                            
  |=============================================                         |  64%
  |                                                                            
  |=============================================                         |  65%
  |                                                                            
  |==============================================                        |  66%
  |                                                                            
  |===============================================                       |  67%
  |                                                                            
  |================================================                      |  68%
  |                                                                            
  |================================================                      |  69%
  |                                                                            
  |=================================================                     |  70%
  |                                                                            
  |==================================================                    |  71%
  |                                                                            
  |==================================================                    |  72%
  |                                                                            
  |===================================================                   |  73%
  |                                                                            
  |====================================================                  |  74%
  |                                                                            
  |=====================================================                 |  75%
  |                                                                            
  |=====================================================                 |  76%
  |                                                                            
  |======================================================                |  77%
  |                                                                            
  |======================================================                |  78%
  |                                                                            
  |=======================================================               |  78%
  |                                                                            
  |=======================================================               |  79%
  |                                                                            
  |========================================================              |  80%
  |                                                                            
  |=========================================================             |  81%
  |                                                                            
  |=========================================================             |  82%
  |                                                                            
  |==========================================================            |  82%
  |                                                                            
  |==========================================================            |  83%
  |                                                                            
  |===========================================================           |  84%
  |                                                                            
  |===========================================================           |  85%
  |                                                                            
  |============================================================          |  86%
  |                                                                            
  |=============================================================         |  87%
  |                                                                            
  |==============================================================        |  88%
  |                                                                            
  |==============================================================        |  89%
  |                                                                            
  |===============================================================       |  90%
  |                                                                            
  |================================================================      |  91%
  |                                                                            
  |================================================================      |  92%
  |                                                                            
  |=================================================================     |  93%
  |                                                                            
  |==================================================================    |  94%
  |                                                                            
  |===================================================================   |  95%
  |                                                                            
  |===================================================================   |  96%
  |                                                                            
  |====================================================================  |  97%
  |                                                                            
  |====================================================================  |  98%
  |                                                                            
  |===================================================================== |  98%
  |                                                                            
  |===================================================================== |  99%
  |                                                                            
  |======================================================================| 100%
#> 
  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |===                                                                   |   4%
  |                                                                            
  |=====                                                                 |   8%
  |                                                                            
  |========                                                              |  12%
  |                                                                            
  |===========                                                           |  15%
  |                                                                            
  |=============                                                         |  19%
  |                                                                            
  |================                                                      |  23%
  |                                                                            
  |===================                                                   |  27%
  |                                                                            
  |======================                                                |  31%
  |                                                                            
  |========================                                              |  35%
  |                                                                            
  |===========================                                           |  38%
  |                                                                            
  |==============================                                        |  42%
  |                                                                            
  |================================                                      |  46%
  |                                                                            
  |===================================                                   |  50%
  |                                                                            
  |======================================                                |  54%
  |                                                                            
  |========================================                              |  58%
  |                                                                            
  |===========================================                           |  62%
  |                                                                            
  |==============================================                        |  65%
  |                                                                            
  |================================================                      |  69%
  |                                                                            
  |===================================================                   |  73%
  |                                                                            
  |======================================================                |  77%
  |                                                                            
  |=========================================================             |  81%
  |                                                                            
  |===========================================================           |  85%
  |                                                                            
  |==============================================================        |  88%
  |                                                                            
  |=================================================================     |  92%
  |                                                                            
  |===================================================================   |  96%
  |                                                                            
  |======================================================================| 100%

head(MSstatsPTM.summary$PTM$ProteinLevelData)
#>   RUN     Protein LogIntensities originalRUN GROUP SUBJECT
#> 1   3 Q9UHD8_K028       20.40683   CCCP-B2T1  CCCP    BCH2
#> 2   4 Q9UHD8_K028       20.42412   CCCP-B2T2  CCCP    BCH2
#> 3   7 Q9UHD8_K028       20.62455  Combo-B2T1 Combo    BCH2
#> 4   8 Q9UHD8_K028       20.72569  Combo-B2T2 Combo    BCH2
#> 5  11 Q9UHD8_K028       20.40666   Ctrl-B2T1  Ctrl    BCH2
#> 6  12 Q9UHD8_K028       20.65381   Ctrl-B2T2  Ctrl    BCH2
#>   TotalGroupMeasurements NumMeasuredFeature MissingPercentage more50missing
#> 1                      4                  1                 0         FALSE
#> 2                      4                  1                 0         FALSE
#> 3                      4                  1                 0         FALSE
#> 4                      4                  1                 0         FALSE
#> 5                      4                  1                 0         FALSE
#> 6                      4                  1                 0         FALSE
#>   NumImputedFeature
#> 1                 0
#> 2                 0
#> 3                 0
#> 4                 0
#> 5                 0
#> 6                 0
head(MSstatsPTM.summary$PROTEIN$ProteinLevelData)
#>   RUN Protein LogIntensities   originalRUN    GROUP SUBJECT
#> 1   3  Q9UHD8       19.36883     CCCP-B2T1     CCCP    BCH2
#> 2   4  Q9UHD8       19.56289     CCCP-B2T2     CCCP    BCH2
#> 3   7  Q9UHD8       18.69612    Combo-B2T1    Combo    BCH2
#> 4  11  Q9UHD8       19.77119     Ctrl-B2T1     Ctrl    BCH2
#> 5  12  Q9UHD8       19.62490     Ctrl-B2T2     Ctrl    BCH2
#> 6  15  Q9UHD8       19.16970 USP30_OE-B2T1 USP30_OE    BCH2
#>   TotalGroupMeasurements NumMeasuredFeature MissingPercentage more50missing
#> 1                      4                  1                 0         FALSE
#> 2                      4                  1                 0         FALSE
#> 3                      4                  1                 0         FALSE
#> 4                      4                  1                 0         FALSE
#> 5                      4                  1                 0         FALSE
#> 6                      4                  1                 0         FALSE
#>   NumImputedFeature
#> 1                 0
#> 2                 0
#> 3                 0
#> 4                 0
#> 5                 0
#> 6                 0

The summarize function returns a list with PTM and Protein summarization information. Each PTM and Protein include a list of data.tables: FeatureLevelData is a data.table of reformatted input of dataSummarizationPTM, ProteinLevelData is the run level summarization data.

1.2.1 QCPlot

Once summarized, MSstatsPTM provides multiple plots to analyze the experiment. Here we show the quality control boxplot. The first plot shows the modified data and the second plot shows the global protein dataset.

1.2.2 Profile Plot

Here we show a profile plot. Again the top plot shows the modified peptide, and the bottom shows the overall protein.

1.3 Modeling - groupComparisonPTM

After summarization, the summarized datasets can be modeled using the groupComparisonPTM function. This function will model the PTM and Protein summarized datasets, and then adjust the PTM model for changes in overall protein abundance. The output of the function is a list containing these three models named: PTM.Model, PROTEIN.Model, ADJUSTED.Model.


# Specify contrast matrix
comparison <- matrix(c(-1,0,1,0),nrow=1)
row.names(comparison) <- "CCCP-Ctrl"
colnames(comparison) <- c("CCCP", "Combo", "Ctrl", "USP30_OE")

MSstatsPTM.model <- groupComparisonPTM(MSstatsPTM.summary, 
                                       data.type = "LabelFree",
                                       contrast.matrix = comparison,
                                       verbose = FALSE)
#> 
  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |=                                                                     |   1%
  |                                                                            
  |=                                                                     |   2%
  |                                                                            
  |==                                                                    |   2%
  |                                                                            
  |==                                                                    |   3%
  |                                                                            
  |===                                                                   |   4%
  |                                                                            
  |===                                                                   |   5%
  |                                                                            
  |====                                                                  |   6%
  |                                                                            
  |=====                                                                 |   7%
  |                                                                            
  |======                                                                |   8%
  |                                                                            
  |======                                                                |   9%
  |                                                                            
  |=======                                                               |  10%
  |                                                                            
  |========                                                              |  11%
  |                                                                            
  |========                                                              |  12%
  |                                                                            
  |=========                                                             |  13%
  |                                                                            
  |==========                                                            |  14%
  |                                                                            
  |===========                                                           |  15%
  |                                                                            
  |===========                                                           |  16%
  |                                                                            
  |============                                                          |  17%
  |                                                                            
  |============                                                          |  18%
  |                                                                            
  |=============                                                         |  18%
  |                                                                            
  |=============                                                         |  19%
  |                                                                            
  |==============                                                        |  20%
  |                                                                            
  |===============                                                       |  21%
  |                                                                            
  |===============                                                       |  22%
  |                                                                            
  |================                                                      |  22%
  |                                                                            
  |================                                                      |  23%
  |                                                                            
  |=================                                                     |  24%
  |                                                                            
  |=================                                                     |  25%
  |                                                                            
  |==================                                                    |  26%
  |                                                                            
  |===================                                                   |  27%
  |                                                                            
  |====================                                                  |  28%
  |                                                                            
  |====================                                                  |  29%
  |                                                                            
  |=====================                                                 |  30%
  |                                                                            
  |======================                                                |  31%
  |                                                                            
  |======================                                                |  32%
  |                                                                            
  |=======================                                               |  33%
  |                                                                            
  |========================                                              |  34%
  |                                                                            
  |=========================                                             |  35%
  |                                                                            
  |=========================                                             |  36%
  |                                                                            
  |==========================                                            |  37%
  |                                                                            
  |==========================                                            |  38%
  |                                                                            
  |===========================                                           |  38%
  |                                                                            
  |===========================                                           |  39%
  |                                                                            
  |============================                                          |  40%
  |                                                                            
  |=============================                                         |  41%
  |                                                                            
  |=============================                                         |  42%
  |                                                                            
  |==============================                                        |  42%
  |                                                                            
  |==============================                                        |  43%
  |                                                                            
  |===============================                                       |  44%
  |                                                                            
  |===============================                                       |  45%
  |                                                                            
  |================================                                      |  46%
  |                                                                            
  |=================================                                     |  47%
  |                                                                            
  |==================================                                    |  48%
  |                                                                            
  |==================================                                    |  49%
  |                                                                            
  |===================================                                   |  50%
  |                                                                            
  |====================================                                  |  51%
  |                                                                            
  |====================================                                  |  52%
  |                                                                            
  |=====================================                                 |  53%
  |                                                                            
  |======================================                                |  54%
  |                                                                            
  |=======================================                               |  55%
  |                                                                            
  |=======================================                               |  56%
  |                                                                            
  |========================================                              |  57%
  |                                                                            
  |========================================                              |  58%
  |                                                                            
  |=========================================                             |  58%
  |                                                                            
  |=========================================                             |  59%
  |                                                                            
  |==========================================                            |  60%
  |                                                                            
  |===========================================                           |  61%
  |                                                                            
  |===========================================                           |  62%
  |                                                                            
  |============================================                          |  62%
  |                                                                            
  |============================================                          |  63%
  |                                                                            
  |=============================================                         |  64%
  |                                                                            
  |=============================================                         |  65%
  |                                                                            
  |==============================================                        |  66%
  |                                                                            
  |===============================================                       |  67%
  |                                                                            
  |================================================                      |  68%
  |                                                                            
  |================================================                      |  69%
  |                                                                            
  |=================================================                     |  70%
  |                                                                            
  |==================================================                    |  71%
  |                                                                            
  |==================================================                    |  72%
  |                                                                            
  |===================================================                   |  73%
  |                                                                            
  |====================================================                  |  74%
  |                                                                            
  |=====================================================                 |  75%
  |                                                                            
  |=====================================================                 |  76%
  |                                                                            
  |======================================================                |  77%
  |                                                                            
  |======================================================                |  78%
  |                                                                            
  |=======================================================               |  78%
  |                                                                            
  |=======================================================               |  79%
  |                                                                            
  |========================================================              |  80%
  |                                                                            
  |=========================================================             |  81%
  |                                                                            
  |=========================================================             |  82%
  |                                                                            
  |==========================================================            |  82%
  |                                                                            
  |==========================================================            |  83%
  |                                                                            
  |===========================================================           |  84%
  |                                                                            
  |===========================================================           |  85%
  |                                                                            
  |============================================================          |  86%
  |                                                                            
  |=============================================================         |  87%
  |                                                                            
  |==============================================================        |  88%
  |                                                                            
  |==============================================================        |  89%
  |                                                                            
  |===============================================================       |  90%
  |                                                                            
  |================================================================      |  91%
  |                                                                            
  |================================================================      |  92%
  |                                                                            
  |=================================================================     |  93%
  |                                                                            
  |==================================================================    |  94%
  |                                                                            
  |===================================================================   |  95%
  |                                                                            
  |===================================================================   |  96%
  |                                                                            
  |====================================================================  |  97%
  |                                                                            
  |====================================================================  |  98%
  |                                                                            
  |===================================================================== |  98%
  |                                                                            
  |===================================================================== |  99%
  |                                                                            
  |======================================================================| 100%
#> 
  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |===                                                                   |   4%
  |                                                                            
  |=====                                                                 |   8%
  |                                                                            
  |========                                                              |  12%
  |                                                                            
  |===========                                                           |  15%
  |                                                                            
  |=============                                                         |  19%
  |                                                                            
  |================                                                      |  23%
  |                                                                            
  |===================                                                   |  27%
  |                                                                            
  |======================                                                |  31%
  |                                                                            
  |========================                                              |  35%
  |                                                                            
  |===========================                                           |  38%
  |                                                                            
  |==============================                                        |  42%
  |                                                                            
  |================================                                      |  46%
  |                                                                            
  |===================================                                   |  50%
  |                                                                            
  |======================================                                |  54%
  |                                                                            
  |========================================                              |  58%
  |                                                                            
  |===========================================                           |  62%
  |                                                                            
  |==============================================                        |  65%
  |                                                                            
  |================================================                      |  69%
  |                                                                            
  |===================================================                   |  73%
  |                                                                            
  |======================================================                |  77%
  |                                                                            
  |=========================================================             |  81%
  |                                                                            
  |===========================================================           |  85%
  |                                                                            
  |==============================================================        |  88%
  |                                                                            
  |=================================================================     |  92%
  |                                                                            
  |===================================================================   |  96%
  |                                                                            
  |======================================================================| 100%
head(MSstatsPTM.model$PTM.Model)
#>        Protein     Label    log2FC         SE    Tvalue DF    pvalue adj.pvalue
#> 1: Q9UHD8_K028 CCCP-Ctrl 0.1147642 0.09463998 1.2126393  4 0.2919872  0.4201767
#> 2: Q9UHD8_K069 CCCP-Ctrl 0.2688399 0.41750153 0.6439256  8 0.5376428  0.6473658
#> 3: Q9UHD8_K141 CCCP-Ctrl 0.7141059 1.15951976 0.6158635  3 0.5815577  0.6642347
#> 4: Q9UHD8_K262 CCCP-Ctrl 0.3076673 0.41648528 0.7387232  8 0.4811835  0.5976805
#> 5: Q9UHQ9_K046 CCCP-Ctrl 1.0516086 0.63193681 1.6641040  4 0.1714238  0.2889715
#> 6: Q9UHQ9_K062 CCCP-Ctrl 7.4586281 3.91369471 1.9057767  4 0.1293742  0.2336522
#>    issue MissingPercentage ImputationPercentage
#> 1:  <NA>               0.5                    0
#> 2:  <NA>               0.0                    0
#> 3:  <NA>               0.5                    0
#> 4:  <NA>               0.0                    0
#> 5:  <NA>               0.5                    0
#> 6:  <NA>               0.5                    0
head(MSstatsPTM.model$PROTEIN.Model)
#>    Protein     Label     log2FC        SE     Tvalue DF      pvalue adj.pvalue
#> 1:  Q9UHD8 CCCP-Ctrl  0.2321867 0.3054474  0.7601529  3 0.502444586 0.67065761
#> 2:  Q9UHQ9 CCCP-Ctrl -0.1543455 0.1532654 -1.0070472  4 0.370886065 0.64286918
#> 3:  Q9UIA9 CCCP-Ctrl  0.1738736 0.1096855  1.5852005  9 0.147381886 0.33080672
#> 4:  Q9UIF8 CCCP-Ctrl  1.1429060 0.2462052  4.6420872  4 0.009718807 0.06317225
#> 5:  Q9UL25 CCCP-Ctrl -2.0671120 0.2668733 -7.7456678  3 0.004475377 0.03878660
#> 6:  Q9UM54 CCCP-Ctrl -0.3602191 0.4761387 -0.7565424  8 0.471013931 0.67065761
#>    issue MissingPercentage ImputationPercentage
#> 1:    NA         0.5000000            0.0000000
#> 2:    NA         0.5000000            0.0000000
#> 3:    NA         0.2500000            0.2500000
#> 4:    NA         0.5000000            0.0000000
#> 5:    NA         0.5000000            0.0000000
#> 6:    NA         0.3333333            0.3333333
head(MSstatsPTM.model$ADJUSTED.Model)
#>        Protein     Label      log2FC        SE      Tvalue        DF    pvalue
#> 1: Q9UHD8_K028 CCCP-Ctrl -0.11742259 0.3197731 -0.36720591  3.578917 0.7341316
#> 2: Q9UHD8_K069 CCCP-Ctrl  0.03665317 0.5173062  0.07085392 10.689428 0.9448222
#> 3: Q9UHD8_K141 CCCP-Ctrl  0.48191914 1.1990764  0.40190862  3.414364 0.7116107
#> 4: Q9UHD8_K262 CCCP-Ctrl  0.07548059 0.5164863  0.14614248 10.680564 0.8865306
#> 5: Q9UHQ9_K046 CCCP-Ctrl  1.20595408 0.6502572  1.85458014  4.468955 0.1297166
#> 6: Q9UHQ9_K062 CCCP-Ctrl  7.61297362 3.9166946  1.94372408  4.012269 0.1236293
#>    adj.pvalue GlobalProtein
#> 1:  0.8250241        Q9UHD8
#> 2:  0.9694697        Q9UHD8
#> 3:  0.8074044        Q9UHD8
#> 4:  0.9176370        Q9UHD8
#> 5:  0.2319176        Q9UHQ9
#> 6:  0.2244347        Q9UHQ9

1.3.1 Volcano Plot

The models from the groupComparisonPTM function can be used in the model visualization function, groupComparisonPlotsPTM. Here we show Volcano Plots for the models.

1.3.2 Heatmap Plot

Here we show a Heatmap for the models.