---
title: "MSstatsTMT : A package for protein significance analysis in shotgun mass spectrometry-based proteomic experiments with tandem mass tag (TMT) labeling"
author: "Ting Huang (<thuang0703@gmail.com>), Meena Choi (<mnchoi67@gmail.com>), Sicheng Hao (<hao.sic@husky.neu.edu>), Olga Vitek(<o.vitek@northeastern.edu>)"
date: "`r Sys.Date()`"
output: rmarkdown::html_vignette
vignette: >
  %\VignetteIndexEntry{MSstatsTMT User Guide}
  %\VignetteEngine{knitr::rmarkdown}
  %\VignetteEncoding{UTF-8}
---

```{r setup, include = FALSE}
knitr::opts_chunk$set(
  collapse = TRUE,
  comment = "#>"
)
```

```{r}
library(MSstatsTMT)
```


This vignette summarizes the introduction and various options of all functionalities in MSstatsTMT. 

- A set of tools for detecting differentially abundant peptides and proteins in shotgun mass spectrometry-based proteomic experiments with tandem mass tag (TMT) labeling.
- The types of experiment that MSstatsTMT supports for metabolic labeling or iTRAQ experiments. LC-MS, SRM, DIA(SWATH) with label-free or labeled synthetic peptides can be analyzed with other R package, MSstats.

MSstatsTMT includes the following three steps for statistical testing: 

1. Converters for different peptide quantification tools to get the input with required format: `PDtoMSstatsTMTFormat`, `MaxQtoMSstatsTMTFormat`, `SpectroMinetoMSstatsTMTFormat` and `OpenMStoMSstatsTMTFormat`.
2. Protein summarization based on peptide quantification data: `proteinSummarization`
3. Group comparison on protein quantification data:  `groupComparisonTMT`


## 1. Converters for different peptide quantification tools

### PDtoMSstatsTMTFormat()

Preprocess PSM data from Proteome Discoverer and convert into the required input format for MSstatsTMT.


#### Arguments

* `input` : data name of Proteome discover PSM output. Read PSM sheet.
* `annotation` : data frame which contains column `Run`, `Fraction`, `TechRepMixture`, `Channel`, `Condition`, `BioReplicate`, `Mixture`.
* `which.proteinid` : Use `Protein.Accessions`(default) column for protein name. `Master.Protein.Accessions` can be used instead.
* `useNumProteinsColumn` : TURE(default) remove shared peptides by information of # Proteins column in PSM sheet.
* `useUniquePeptide` : TRUE(default) removes peptides that are assigned for more than one proteins. We assume to use unique peptide for each protein.
* `rmPSM_withMissing_withinRun` : TRUE will remove PSM with any missing value within each Run. Default is FALSE.
* `rmPSM_withfewMea_withinRun` : only for rmPSM_withMissing_withinRun = FALSE. TRUE(default) will remove the features that have 1 or 2 measurements within each Run.
* `removeProtein_with1Peptide` : TRUE will remove the proteins which have only 1 peptide and charge. Default is FALSE.
* `summaryforMultipleRows` : sum(default) or max - when there are multiple measurements for certain PSM in certain run, select the PSM with the largest summation or maximal value.


#### Example
```{r}
# read in PD PSM sheet
# raw.pd <- read.delim("161117_SILAC_HeLa_UPS1_TMT10_5Mixtures_3TechRep_UPSdB_Multiconsensus_PD22_Intensity_PSMs.txt")
head(raw.pd)

# Read in annotation including condition and biological replicates per run and channel.
# Users should make this annotation file. It is not the output from Proteome Discoverer.
# annotation.pd <- read.csv(file="PD_Annotation.csv", header=TRUE)
head(annotation.pd)

# do not remove PSM with missing values within one run
input.pd <- PDtoMSstatsTMTFormat(raw.pd, annotation.pd)
head(input.pd)

# remove PSM with missing values within one run
input.pd.no.miss <- PDtoMSstatsTMTFormat(raw.pd, annotation.pd,
                                 rmPSM_withMissing_withinRun = TRUE)
head(input.pd.no.miss)
```

### MaxQtoMSstatsTMTFormat()

Preprocess PSM-level data from MaxQuant and convert into the required input format for MSstatsTMT.


#### Arguments

* `evidence` : name of `evidence.txt` data, which includes PSM-level data.
* `proteinGroups` : name of `proteinGroups.txt` data, which contains the detailed information of protein identifications.
* `annotation` : data frame which contains column `Run`, `Fraction`, `TechRepMixture`, `Channel`, `Condition`, `BioReplicate`, `Mixture`.
* `which.proteinid` : Use `Proteins`(default) column for protein name. `Leading.proteins` or `Leading.razor.proteins` can be used instead. However, those can potentially have the shared peptides.
* `rmProt_Only.identified.by.site` : TRUE will remove proteins with '+' in 'Only.identified.by.site' column from proteinGroups.txt, which was identified only by a modification site. FALSE is the default.
* `useUniquePeptide` : TRUE(default) removes peptides that are assigned for more than one proteins. We assume to use unique peptide for each protein.
* `rmPSM_withMissing_withinRun` : TRUE will remove PSM with any missing value within each Run. Default is FALSE.
* `rmPSM_withfewMea_withinRun` : only for rmPSM_withMissing_withinRun = FALSE. TRUE(default) will remove the features that have 1 or 2 measurements within each Run.
* `removeProtein_with1Peptide` : TRUE will remove the proteins which have only 1 peptide and charge. Default is FALSE.
* `summaryforMultipleRows` : sum(default) or max - when there are multiple measurements for certain PSM in certain run, select the PSM with the largest summation or maximal value.


#### Example
```{r}
# Read in MaxQuant files
# proteinGroups <- read.table("proteinGroups.txt", sep="\t", header=TRUE)

# evidence <- read.table("evidence.txt", sep="\t", header=TRUE)

# Users should make this annotation file. It is not the output from MaxQuant.
# annotation.mq <- read.csv(file="MQ_Annotation.csv", header=TRUE)

input.mq <- MaxQtoMSstatsTMTFormat(evidence, proteinGroups, annotation.mq)
head(input.mq)
```

### SpectroMinetoMSstatsTMTFormat()

Preprocess PSM data from SpectroMine and convert into the required input format for MSstatsTMT.


#### Arguments

* `input` : data name of SpectroMine PSM output. Read PSM sheet.
* `annotation` : data frame which contains column `Run`, `Fraction`, `TechRepMixture`, `Channel`, `Condition`, `BioReplicate`, `Mixture`.
* `filter_with_Qvalue` : TRUE(default) will filter out the intensities that have greater than qvalue_cutoff in EG.Qvalue column. Those intensities will be replaced with NA and will be considered as censored missing values for imputation purpose.
* `qvalue_cutoff` : Cutoff for EG.Qvalue. default is 0.01.
* `useUniquePeptide` : TRUE(default) removes peptides that are assigned for more than one proteins. We assume to use unique peptide for each protein.
* `rmPSM_withMissing_withinRun` : TRUE will remove PSM with any missing value within each Run. Default is FALSE.
* `rmPSM_withfewMea_withinRun` : only for `rmPSM_withMissing_withinRun = FALSE`. TRUE(default) will remove the features that have 1 or 2 measurements within each Run.
* `removeProtein_with1Peptide` : TRUE will remove the proteins which have only 1 peptide and charge. Default is FALSE.
* `summaryforMultipleRows` : sum(default) or max - when there are multiple measurements for certain PSM in certain run, select the PSM with the largest summation or maximal value.
* `remove_norm_channel` : TRUE(default) removes `Norm` channels from protein level data.
* `remove_empty_channel` : TRUE(default) removes `Empty` channels from protein level data.


#### Example
```{r}
# Read in SpectroMine PSM report
# raw.mine <- read.csv('20180831_095547_CID-OT-MS3-Short_PSM Report_20180831_103118.xls', sep="\t")

# Users should make this annotation file. It is not the output from SpectroMine
# annotation.mine <- read.csv(file="Mine_Annotation.csv", header=TRUE)

input.mine <- SpectroMinetoMSstatsTMTFormat(raw.mine, annotation.mine)
head(input.mine)
```

### OpenMStoMSstatsTMTFormat()

Preprocess MSstatsTMT report from OpenMS and convert into the required input format for MSstatsTMT.


#### Arguments

* `input`: data name of MSstatsTMT report from OpenMS. Read csv file.
* `useUniquePeptide` : TRUE(default) removes peptides that are assigned for more than one proteins. We assume to use unique peptide for each protein.
* `rmPSM_withMissing_withinRun` : TRUE will remove PSM with any missing value within each Run. Default is FALSE.
* `rmPSM_withfewMea_withinRun` : only for rmPSM_withMissing_withinRun = FALSE. TRUE(default) will remove the features that have 1 or 2 measurements within each Run.
* `removeProtein_with1Peptide` : TRUE will remove the proteins which have only 1 peptide and charge. Default is FALSE.
* `summaryforMultipleRows` : sum(default) or max - when there are multiple measurements for certain PSM in certain run, select the PSM with the largest summation or maximal value.

#### Example
```{r}
# read in MSstatsTMT report from OpenMS
# raw.om <- read.csv("OpenMS_20200222/20200225_MSstatsTMT_OpenMS_Export.csv")
head(raw.om)

# the function only requries one input file
input.om <- OpenMStoMSstatsTMTFormat(raw.om)
head(input.om)
```

## 2. Protein summarization, normalization and visualization

### 2.1. proteinSummarization()

Global median normalization is first applied to peptide level quantification data (equalizing the medians across all the channels and MS runs). Protein-level summarization from peptide level quantification should be performed before testing differentially abundant proteins. Then, normalization between MS runs using normalization channels will be implemented. In particular, protein summarization method `MSstats` assumes missing values are censored and then imputes the missing values before summarizing peptide level data into protein level data. Other methods, including `MedianPolish`, `Median` and `LogSum`, do not impute missing values.

#### Arguments

* `data` : Name of the output of PDtoMSstatsTMTFormat function or peptide-level quantified data from other tools. It should have columns named `Protein`, `PSM`, `TechRepMixture`, `Mixture`, `Run`, `Channel`, `Condition`, `BioReplicate`, `Intensity`.
* `method` : Four different summarization methods to protein-level can be performed : `msstats`(default), `MedianPolish`, `Median`, `LogSum`.
* `global_norm` :  Global median normalization on peptide level data (equalizing the medians across all the channels and MS runs). Default is TRUE. It will be performed before protein-level summarization.
* `reference_norm` :  Reference channel based normalization between MS runs. TRUE(default) needs at least one reference channel in each MS run, annotated by `Norm` in Condtion column. It will be performed after protein-level summarization. FALSE will not perform this normalization step. If data only has one run, then reference_norm=FALSE.
* `remove_norm_channel` :  TRUE(default) removes `Norm` channels from protein level data.
* `remove_empty_channel` :  TRUE(default) removes `Empty` channels from protein level data.
* `MBimpute` : only for `method = "msstats"`. TRUE (default) imputes missing values by Accelated failure model. FALSE uses minimum value to impute the missing value for each peptide precursor ion.
* `maxQuantileforCensored` : We assume missing values are censored. `maxQuantileforCensored` is Maximum quantile for deciding censored missing value, for instance, 0.999. Default is Null.

#### Example
```{r message = FALSE , warning = FALSE}
# use MSstats for protein summarization
quant.msstats <- proteinSummarization(input.pd,
                                      method="msstats",
                                      global_norm=TRUE,
                                      reference_norm=TRUE,
                                      remove_norm_channel = TRUE,
                                      remove_empty_channel = TRUE)
head(quant.msstats)

# use Median for protein summarization
# since median method doesn't impute missing values, 
# we need to use the input data without missing values
quant.median <- proteinSummarization(input.pd.no.miss,
                                     method="Median",
                                     global_norm=TRUE,
                                     reference_norm=TRUE,
                                     remove_norm_channel = TRUE,
                                     remove_empty_channel = TRUE)

head(quant.median)
```

### 2.2 dataProcessPlotsTMT()

Visualization for explanatory data analysis. To illustrate the quantitative data after data-preprocessing and quality control of TMT runs, dataProcessPlotsTMT takes the quantitative data from converter functions (`PDtoMSstatsTMTFormat`, `MQtoMSstatsTMTFormat` and `SpectroMinetoMSstatsTMTFormat`) and summarized data from function `proteinSummarization` as input. It generates two types of figures in pdf files as output :

(1) profile plot (specify "ProfilePlot" in option type), to identify the potential sources of variation for each protein;

(2) quality control plot (specify "QCPlot" in option type), to evaluate the systematic bias between MS runs.


#### Arguments

* `data.peptide` : name of the data with peptide-level, which can be the output of converter functions (`PDtoMSstatsTMTFormat`, `MQtoMSstatsTMTFormat` and `SpectroMinetoMSstatsTMTFormat`).
* `data.summarization` : name of the data with protein-level, which can be the output of `proteinSummarization` function.
* `type` : choice of visualization. "ProfilePlot" represents profile plot of log intensities across MS runs.
"QCPlot" represents quality control plot of log intensities across MS runs.
* `ylimUp` : upper limit for y-axis in the log scale.
FALSE(Default) for Profile Plot and QC Plot use the upper limit as rounded off maximum of log2(intensities) after normalization + 3.
* `ylimDown` : lower limit for y-axis in the log scale. FALSE(Default) for Profile Plot and QC Plot is 0.
* `x.axis.size` : size of x-axis labeling for "Run" and "channel" in Profile Plot and QC Plot.
* `y.axis.size` : size of y-axis labels. Default is 10.
* `text.size` : size of labels represented each condition at the top of graph in Profile Plot and QC plot. Default is 4.
* `text.angle` : angle of labels represented each condition at the top of graph in Profile Plot and QC plot. Default is 0.
* `legend.size` : size of legend above graph in Profile Plot. Default is 7.
* `dot.size.profile` : size of dots in profile plot. Default is 2.
* `ncol.guide` : number of columns for legends at the top of plot. Default is 5.
* `width` : width of the saved file. Default is 10.
* `height` : height of the saved file. Default is 10.
* `which.Protein` : Protein list to draw plots. List can be names of Proteins or order numbers of Proteins.
Default is "all", which generates all plots for each protein. For QC plot, "allonly" will generate one QC plot with all proteins.
* `originalPlot` : TRUE(default) draws original profile plots, without normalization.
* `summaryPlot` : TRUE(default) draws profile plots with protein summarization for each channel and MS run.
* `address` : the name of folder that will store the results. Default folder is the current working directory.
The other assigned folder has to be existed under the current working directory.
An output pdf file is automatically created with the default name of "ProfilePlot.pdf" or "QCplot.pdf".
The command address can help to specify where to store the file as well as how to modify the beginning of the file name.
If address=FALSE, plot will be not saved as pdf file but showed in window.

#### Example
```{r}
## Profile plot without norm channnels and empty channels
dataProcessPlotsTMT(data.peptide = input.pd,
                     data.summarization = quant.msstats,
                     type = 'ProfilePlot',
                     width = 21, # adjust the figure width since there are 15 TMT runs.
                     height = 7)

# ## Profile plot with all the channels
# quant.msstats.all <- proteinSummarization(input.pd,
#                                       method="msstats",
#                                       normalization=TRUE,
#                                       remove_norm_channel=FALSE,
#                                       remove_empty_channel=FALSE)
# 
# dataProcessPlotsTMT(data.peptide = input.pd,
#                      data.summarization = quant.msstats.all,
#                      type = 'ProfilePlot',
#                      width = 21, # adjust the figure width since there are 15 TMT runs.
#                      height = 7)

## Quality control plot 
# dataProcessPlotsTMT(data.peptide=input.pd,
                     # data.summarization=quant.msstats, 
                     # type='QCPlot',
                     # width = 21, # adjust the figure width since there are 15 TMT runs. 
                     # height = 7)
```

## 3. groupComparisonTMT()

Tests for significant changes in protein abundance across conditions based on a family of linear mixed-effects models in TMT experiment. Experimental design of case-control study (patients are not repeatedly measured) is automatically determined based on proper statistical model.

### Arguments

* `data` : Name of the output of proteinSummarization function. It should have columns named `Protein`, `TechRepMixture`,  `Mixture`, `Run`, `Channel`, `Condition`, `BioReplicate`, `Abundance`.
* `contrast.matrix` : Comparison between conditions of interests. 1) default is `pairwise`, which compare all possible pairs between two conditions. 2) Otherwise, users can specify the comparisons of interest. Based on the levels of conditions, specify 1 or -1 to the conditions of interests and 0 otherwise. The levels of conditions are sorted alphabetically.
* `moderated` : If moderated = TRUE, then moderated t statistic will be calculated; otherwise, ordinary t statistic will be used.
* `adj.method` : adjusted method for multiple comparison. 'BH` is default.


### Example
```{r message = FALSE, warning = FALSE}
# test for all the possible pairs of conditions
test.pairwise <- groupComparisonTMT(quant.msstats)
head(test.pairwise)

# Check the conditions in the protein data
levels(quant.msstats$Condition)
# Only compare condition 0.125 and 1
comparison<-matrix(c(-1,0,0,1),nrow=1)
# Set the names of each row
row.names(comparison)<-"1-0.125"
# Set the column names
colnames(comparison)<- c("0.125", "0.5", "0.667", "1")
comparison

test.contrast <- groupComparisonTMT(data = quant.msstats, contrast.matrix = comparison)
head(test.contrast)
```
