Package ‘MSstats’

November 6, 2018
Title Protein Significance Analysis in DDA, SRM and DIA for Label-free
or Label-based Proteomics Experiments
Version 3.14.0
Date 2018-07-30
Description A set of tools for statistical relative protein significance analy-
sis in DDA, SRM and DIA experiments.
Maintainer Meena Choi <[email protected]>
License Artistic-2.0
Depends R (>= 3.4)
Imports lme4, marray, limma, gplots, ggplot2, methods, grid, ggrepel,
preprocessCore, reshape2, survival, minpack.lm, utils,
grDevices, graphics, stats, doSNOW, snow, foreach, data.table,
MASS, dplyr, tidyr, stringr, randomForest
Suggests BiocStyle, knitr, rmarkdown, MSstatsBioData
VignetteBuilder knitr
biocViews MassSpectrometry, Proteomics, Software, Normalization,
QualityControl, TimeCourse
LazyData true

URL http://msstats.org

BugReports https://groups.google.com/forum/#!forum/msstats
RoxygenNote 6.0.1
NeedsCompilation no
Author Meena Choi [aut, cre],
Cyril Galitzine [aut],
Ting Huang [aut],
Tsung-Heng Tsai [aut],
Olga Vitek [aut]
git_url https://git.bioconductor.org/packages/MSstats
git_branch RELEASE_3_8
git_last_commit 03ea7c1
git_last_commit_date 2018-10-30
Date/Publication 2018-11-05

1
2 MSstats-package

R topics documented:
MSstats-package . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
dataProcess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
dataProcessPlots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
DDARawData . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
DDARawData.Skyline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
designSampleSize . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
designSampleSizeClassification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
designSampleSizeClassificationPlots . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
designSampleSizePlots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
DIARawData . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
DIAUmpiretoMSstatsFormat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
groupComparison . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
groupComparisonPlots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
linear_quantlim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
MaxQtoMSstatsFormat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
modelBasedQCPlots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
nonlinear_quantlim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
OpenMStoMSstatsFormat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
OpenSWATHtoMSstatsFormat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
PDtoMSstatsFormat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
plot_quantlim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
ProgenesistoMSstatsFormat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
SkylinetoMSstatsFormat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
SpectronauttoMSstatsFormat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
SpikeInDataLinear . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
SpikeInDataNonLinear . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
SRMRawData . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Index 47

MSstats-package Tools for protein significance analysis in DDA,SRM and DIA pro-
teomic experiments for label-free workflows or workflows with stable
isotope labeled reference

Description
A set of tools for protein significance analysis in SRM, DDA and DIA experiments.

Details

Package: MSstats
License: Artistic-2.0
LazyLoad: yes

The package includes four main sections: I. explanatory data analysis (data pre-processing and
quality control of MS runs), II. model-based analysis (finding differentially abundant proteins), III.
dataProcess 3

statistical design of future experiments (sample size calculations), and IV. protein quantification
(estimation of protein abundance). Section I contains functions for (1) data pre-processing and
quality control of MS runs (see dataProcess) and (2) visualizing for explanatory data analysis (see
dataProcessPlots). Section II contains functions for (1) finding differentially abundant proteins
(see groupComparison) and (2) visualizing for the testing results (see groupComparisonPlots)
and for checking normality assumption (see modelBasedQCPlots). Section III contains functions
for (1) calculating sample size (see designSampleSize) and (2) visualizing for the sample size cal-
culations (see designSampleSizePlots). Section IV contains functions for (1) per-protein group
quantification and patient quantification (see quantification)
Examples of data in MSstats are (1) example of required input data format from label-based SRM
experiment SRMRawData; (2) example of required input data format from DDA experiment DDARawData;
(3) example of required input data format from label-free SWATH experiment DIARawData.
The functions for converting the output from spectral processing tools, (1) Skyline, SkylinetoMSstatsFormat,
(2) MaxQuant, MaxQtoMSstatsFormat, (3) Progenesis, ProgenesistoMSstatsFormat, (4) Spec-
tronaut, SpectronauttoMSstatsFormat, (5) Proteome discovere, PDtoMSstatsFormat, (6) OpenMS,
OpenMStoMSstatsFormat, (7) OpenSWATH, OpenSWATHtoMSstatsFormat, and (8) DIAUmpire,
DIAUmpiretoMSstatsFormatare available.

Author(s)

Meena Choi, Cyril Galitzine, Tsung-Heng Tsai, Olga Vitek.

Maintainer: Meena Choi (<[email protected]>)

References

Meena Choi, Ching-Yun Chang, Timothy Clough, Daniel Broudy, Trevor Killeen, Brendan MacLean
and Olga Vitek. "MSstats: an R package for statistical analysis of quantitative mass spectrometry-
based proteomic experiments" Bioinformatics, 30(17):2524-2526, 2014.
Ching-Yun Chang, Paola Picotti, Ruth Huttenhain, Viola Heinzelmann-Schwarz, Marko Jovanovic,
Ruedi Aebersold, Olga Vitek. "Protein significance analysis in selected reaction monitoring (SRM)
measurements." Molecular & Cellular Proteomics, 11:M111.014662, 2012.
Timothy Clough, Safia Thaminy, Susanne Ragg, Ruedi Aebersold, Olga Vitek. "Statistical protein
quantification and significance analysis in label-free LC-M experiments with complex designs"
BMC Bioinformatics, 13:S16, 2012.

dataProcess Data pre-processing and quality control of MS runs of raw data

Description

Data pre-processing and quality control of MS runs of the original raw data into quantitative data
for model fitting and group comparison. Log transformation is automatically applied and additional
variables are created in columns for model fitting and group comparison process. Three options of
data pre-processing and quality control of MS runs in dataProcess are (1) Transformation: logarithm
transformation with base 2 or 10; (2) Normalization: to remove systematic bias between MS runs.
4 dataProcess

Usage

dataProcess(raw,
logTrans=2,
normalization="equalizeMedians",
nameStandards=NULL,
address="",
fillIncompleteRows=TRUE,
featureSubset="all",
remove_noninformative_feature_outlier=FALSE,
n_top_feature=3,
summaryMethod="TMP",
equalFeatureVar=TRUE,
censoredInt="NA",
cutoffCensored="minFeature",
MBimpute=TRUE,
remove50missing=FALSE,
maxQuantileforCensored=0.999,
clusters=NULL)

Arguments

raw name of the raw (input) data set.

logTrans logarithm transformation with base 2(default) or 10.
normalization normalization to remove systematic bias between MS runs. There are three
different normalizations supported. ’equalizeMedians’(default) represents con-
stant normalization (equalizing the medians) based on reference signals is per-
formed. ’quantile’ represents quantile normalization based on reference signals
is performed. ’globalStandards’ represents normalization with global standards
proteins. FALSE represents no normalization is performed.
nameStandards vector of global standard peptide names. only for normalization with global
standard peptides.
fillIncompleteRows
If the input dataset has incomplete rows, TRUE(default) adds the rows with
intensity value=NA for missing peaks. FALSE reports error message with list
of features which have incomplete rows.
featureSubset "all"(default) uses all features that the data set has. "top3" uses top 3 features
which have highest average of log2(intensity) across runs. "topN" uses top N
features which has highest average of log2(intensity) across runs. It needs the
input for n_top_feature option. "highQuality" is under development. Currently
it will use top 3 features.
remove_noninformative_feature_outlier
It only works with featureSubset="highQuality". TRUE allows to remove 1) the
features with column : feature_quality="Noninformative" which are featurew
with bad quality, 2) outliers that are flagged in the column, is_outlier=TRUE.
FALSE (default) does not allow to remove the proteins, in which all features are
interfered. In this case, the proteins, which will completely loss all features by
the algorithm, will keep the most abundant peptide.
n_top_feature The number of top features for featureSubset=’topN’. Default is 3, which means
to use top 3 features.
dataProcess 5

summaryMethod "TMP"(default) means Tukey’s median polish, which is robust estimation method.
"linear" uses linear mixed model.
equalFeatureVar
only for summaryMethod="linear". default is TRUE. Logical variable for whether
the model should account for heterogeneous variation among intensities from
different features. Default is TRUE, which assume equal variance among inten-
sities from features. FALSE means that we cannot assume equal variance among
intensities from features, then we will account for heterogeneous variation from
different features.
censoredInt Missing values are censored or at random. ’NA’ (default) assumes that all ’NA’s
in ’Intensity’ column are censored. ’0’ uses zero intensities as censored inten-
sity. In this case, NA intensities are missing at random. The output from Skyline
should use ’0’. Null assumes that all NA intensites are randomly missing.
cutoffCensored Cutoff value for censoring. only with censoredInt=’NA’ or ’0’. Default is ’min-
Feature’, which uses minimum value for each feature.’minFeatureNRun’ uses
the smallest between minimum value of corresponding feature and minimum
value of corresponding run. ’minRun’ uses minumum value for each run.
MBimpute only for summaryMethod="TMP" and censoredInt=’NA’ or ’0’. TRUE (de-
fault) imputes ’NA’ or ’0’ (depending on censoredInt option) by Accelated fail-
ure model. FALSE uses the values assigned by cutoffCensored.
remove50missing
only for summaryMethod="TMP". TRUE removes the runs which have more
than 50% missing values. FALSE is default.
address the name of folder that will store the results. Default folder is the current work-
ing directory. The other assigned folder has to be existed under the current
working directory. An output csv file is automatically created with the default
name of "BetweenRunInterferenceFile.csv". The command address can help to
specify where to store the file as well as how to modify the beginning of the file
name.
maxQuantileforCensored
Maximum quantile for deciding censored missing values. default is 0.999
clusters a user specified number of clusters. default is NULL, which does not use cluster.

Details
• raw : See SRMRawData for the required data structure of raw (input) data.
• logTrans : if logTrans=2, the measurement of Variable ABUNDANCE is log-transformed
with base 2. Same apply to logTrans=10.
• normalization : if normalization=TRUE and logTrans=2, the measurement of Variable ABUN-
DANCE is log-transformed with base 2 and normalized. Same as for logTrans=10.
• featureSubset : After the data was normalized, we deeply looked at each single feature (which
is a precursor in DDA, a fragment in DIA, and a transition in SRM) and quantify its un-
explainable variation. Ultimately, we remove the features with interference.
• equalFeatureVar : If the unequal variation of error for different peptide features is detected,
then a possible solution is to account for the unequal error variation by means of a procedure
called iteratively re-weighted least squares. equalFeatureVar=FALSE performs an iterative
fitting procedure, in which features are weighted inversely proportionaly to the variation in
their intensities, so that feature with large variation are given less importance in the estimation
of parameters in the model.
6 dataProcess

Value
A list of data.frame ProcessedData is the data.frame of reformatted input of dataProcess including
extra columns, such as log2-transformed and normalized intensities (abundance column); Runlevel-
Data is the the data.frame for run-level summarized data.

Warning
When a transition is missing completely in a condition or a MS run, a warning message is sent to
the console notifying the user of the missing transitions.
The types of experiment that MSstats can analyze are LC-MS, SRM, DIA(SWATH) with label-free
or labeled synthetic peptides. MSstats does not support for metabolic labeling or iTRAQ experi-
ments.

Author(s)
Meena Choi, Olga Vitek.
Maintainer: Meena Choi (<[email protected]>)

References
Meena Choi, Ching-Yun Chang, Timothy Clough, Daniel Broudy, Trevor Killeen, Brendan MacLean
and Olga Vitek. "MSstats: an R package for statistical analysis of quantitative mass spectrometry-
based proteomic experiments" Bioinformatics, 30(17):2524-2526, 2014.
Ching-Yun Chang, Paola Picotti, Ruth Huttenhain, Viola Heinzelmann-Schwarz, Marko Jovanovic,
Ruedi Aebersold, Olga Vitek. "Protein significance analysis in selected reaction monitoring (SRM)
measurements" Molecular & Cellular Proteomics, 11:M111.014662, 2012.
Timothy Clough, Safia Thaminy, Susanne Ragg, Ruedi Aebersold, Olga Vitek. "Statistical protein
quantification and significance analysis in label-free LC-M experiments with complex designs"
BMC Bioinformatics, 13:S16, 2012.

Examples
# Consider a raw data (i.e. SRMRawData) for a label-based SRM experiment from a yeast study
# with ten time points (T1-T10) of interests and three biological replicates.
# It is a time course experiment. The goal is to detect protein abundance changes
# across time points.

head(SRMRawData)

# Log2 transformation and normalization are applied (default)

QuantData<-dataProcess(SRMRawData)
head(QuantData$ProcessedData)

# Log10 transformation and normalization are applied

QuantData1<-dataProcess(SRMRawData, logTrans=10)
head(QuantData1$ProcessedData)

# Log2 transformation and no normalization are applied

QuantData2<-dataProcess(SRMRawData,normalization=FALSE)
head(QuantData2$ProcessedData)
dataProcessPlots 7

dataProcessPlots Visualization for explanatory data analysis

Description
To illustrate the quantitative data after data-preprocessing and quality control of MS runs, dat-
aProcessPlots takes the quantitative data from function (dataProcess) as input and automatically
generate three 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; (3) mean plot for con-
ditions (specify "ConditionPlot" in option type), to illustrate mean and variability of each condition
per protein.

Usage
dataProcessPlots(data=data,
type=type,
featureName="Transition",
ylimUp=FALSE,
ylimDown=FALSE,
scale=FALSE,
interval="CI",
x.axis.size=10,
y.axis.size=10,
text.size=4,
text.angle=0,
legend.size=7,
dot.size.profile=2,
dot.size.condition=3,
width=10,
height=10,
which.Protein="all",
originalPlot=TRUE,
summaryPlot=TRUE,
save_condition_plot_result=FALSE,
address="")

Arguments
data name of the (output of dataProcess function) data set.
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. "ConditionPlot" represents mean plot of log ratios (Light/Heavy)
across conditions.
featureName for "ProfilePlot" only, "Transition" (default) means printing feature legend in
transition-level; "Peptide" means printing feature legend in peptide-level; "NA"
means no feature legend printing.
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. FALSE(Default) for Condition Plot is maximum of log ratio
+ SD or CI.
8 dataProcessPlots

ylimDown lower limit for y-axis in the log scale. FALSE(Default) for Profile Plot and QC
Plot is 0. FALSE(Default) for Condition Plot is minumum of log ratio - SD or
CI.
scale for "ConditionPlot" only, FALSE(default) means each conditional level is not
scaled at x-axis according to its actual value (equal space at x-axis). TRUE
means each conditional level is scaled at x-axis according to its actual value
(unequal space at x-axis).
interval for "ConditionPlot" only, "CI"(default) uses confidence interval with 0.95 sig-
nificant level for the width of error bar. "SD" uses standard deviation for the
width of error bar.
x.axis.size size of x-axis labeling for "Run" in Profile Plot and QC Plot, and "Condition" in
Condition Plot. Default is 10.
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 or x-axis labeling in Condition plot. Default is 0.
legend.size size of feature legend (transition-level or peptide-level) above graph in Profile
Plot. Default is 7.
dot.size.profile
size of dots in profile plot. Default is 2.
dot.size.condition
size of dots in condition plot. Default is 3.
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 Pro-
teins from levels(data$ProcessedData$PROTEIN). Default is "all", which gen-
erates all plots for each protein. For QC plot, "allonly" will generate one QC
plot with all proteins.
originalPlot TRUE(default) draws original profile plots.
summaryPlot TRUE(default) draws profile plots with summarization for run levels.
save_condition_plot_result
TRUE saves the table with values using condition plots. Default is FALSE.
address the name of folder that will store the results. Default folder is the current work-
ing 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" or "ConditionPlot.pdf" or "Condition-
Plot_value.csv". 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.

Details
• Profile Plot : identify the potential sources of variation of each protein. QuantData$ProcessedData
is used for plots. X-axis is run. Y-axis is log-intensities of transitions. Reference/endogenous
signals are in the left/right panel. Line colors indicate peptides and line types indicate transi-
tions. In summarization plots, gray dots and lines are the same as original profile plots with
QuantData$ProcessedData. Dark dots and lines are for summarized intensities from Quant-
Data$RunlevelData.
dataProcessPlots 9

• QC Plot : illustrate the systematic bias between MS runs. After normalization, the reference
signals for all proteins should be stable across MS runs. QuantData$ProcessedData is used
for plots. X-axis is run. Y-axis is log-intensities of transition. Reference/endogenous signals
are in the left/right panel. The pdf file contains (1) QC plot for all proteins and (2) QC plots
for each protein separately.
• Condition Plot : illustrate the systematic difference between conditions. Summarized in-
tensnties from QuantData$RunlevelData are used for plots. X-axis is condition. Y-axis is
summarized log transformed intensity. If scale is TRUE, the levels of conditions is scaled
according to its actual values at x-axis. Red points indicate the mean for each condition. If
interval is "CI", blue error bars indicate the confidence interval with 0.95 significant level for
each condition. If interval is "SD", blue error bars indicate the standard deviation for each
condition.The interval is not related with model-based analysis.
The input of this function is the quantitative data from function (dataProcess).

Value
pdf will be generated under the working directory.

Author(s)
Meena Choi, Olga Vitek.
Maintainer: Meena Choi (<[email protected]>)

References
Meena Choi, Ching-Yun Chang, Timothy Clough, Daniel Broudy, Trevor Killeen, Brendan MacLean
and Olga Vitek. "MSstats: an R package for statistical analysis of quantitative mass spectrometry-
based proteomic experiments" Bioinformatics, 30(17):2524-2526, 2014.
Ching-Yun Chang, Paola Picotti, Ruth Huttenhain, Viola Heinzelmann-Schwarz, Marko Jovanovic,
Ruedi Aebersold, Olga Vitek. "Protein significance analysis in selected reaction monitoring (SRM)
measurements." Molecular & Cellular Proteomics, 11:M111.014662, 2012.
Timothy Clough, Safia Thaminy, Susanne Ragg, Ruedi Aebersold, Olga Vitek. "Statistical protein
quantification and significance analysis in label-free LC-M experiments with complex designs"
BMC Bioinformatics, 13:S16, 2012.

Examples
# Consider quantitative data (i.e. QuantData) from a yeast study with ten time points of interests,
# three biological replicates, and no technical replicates which is a time-course experiment.
# The goal is to provide pre-analysis visualization by automatically generate two types of figures
# in two separate pdf files.
# Protein IDHC (gene name IDP2) is differentially expressed in time point 1 and time point 7,
# whereas, Protein PMG2 (gene name GPM2) is not.

QuantData<-dataProcess(SRMRawData)
head(QuantData$ProcessedData)

# Profile plot
dataProcessPlots(data=QuantData,type="ProfilePlot")

# Quality control plot

dataProcessPlots(data=QuantData,type="QCPlot")
10 DDARawData

# Quantification plot for conditions

dataProcessPlots(data=QuantData,type="ConditionPlot")

DDARawData Example dataset from a label-free DDA, a controlled spike-in experi-

ment.

Description

This is a data set obtained from a published study (Mueller, et. al, 2007). A controlled spike-in
experiment, where 6 proteins, (horse myoglobin, bovine carbonic anhydrase, horse Cytochrome
C, chicken lysozyme, yeast alcohol dehydrogenase, rabbit aldolase A) were spiked into a complex
background in known concentrations in a latin square design. The experiment contained 6 mixtures,
and each mixture was analyzed in label-free LC-MS mode with 3 technical replicates (resulting
in the total of 18 runs). Each protein was represented by 7-21 peptides, and each peptide was
represented by 1-5 transition.

Usage

DDARawData

Format

data.frame

Details

The raw data (input data for MSstats) is required to contain variable of ProteinName, PeptideSe-
quence, PrecursorCharge, FragmentIon, ProductCharge, IsotopeLabelType, Condition, BioRepli-
cate, Run, Intensity. The variable names should be fixed.
If the information of one or more columns is not available for the original raw data, please retain
the column variables and type in fixed value. For example, the original raw data does not contain
the information of PrecursorCharge and ProductCharge, we retain the column PrecursorCharge and
ProductCharge and then type in NA for all transitions in RawData.
Variable Intensity is required to be original signal without any log transformation and can be speci-
fied as the peak of height or the peak of area under curve.

Value

data.frame with the required format of MSstats.

Author(s)

Meena Choi, Olga Vitek.

Maintainer: Meena Choi (<[email protected]>)
DDARawData.Skyline 11

References
Meena Choi, Ching-Yun Chang, Timothy Clough, Daniel Broudy, Trevor Killeen, Brendan MacLean
and Olga Vitek. "MSstats: an R package for statistical analysis of quantitative mass spectrometry-
based proteomic experiments" Bioinformatics, 30(17):1514-1526, 2014.
Timothy Clough, Safia Thaminy, Susanne Ragg, Ruedi Aebersold, Olga Vitek. "Statistical protein
quantification and significance analysis in label-free LC-M experiments with complex designs"
BMC Bioinformatics, 13:S16, 2012.
Mueller, L. N., Rinner, O., Schmidt, A., Letarte, S., Bodenmiller, B., Brusniak, M., Vitek, O.,
Aebersold, R., and Muller, M. (2007). SuperHirn - a novel tool for high resolution LC-MS based
peptide/protein profiling. Proteomics, 7, 3470-3480. 3, 34

Examples
head(DDARawData)

DDARawData.Skyline Example dataset from a label-free DDA, a controlled spike-in experi-

ment, processed by Skyline.

Description
This is a data set obtained from a published study (Mueller, et. al, 2007). A controlled spike-in
experiment, where 6 proteins, (horse myoglobin, bovine carbonic anhydrase, horse Cytochrome
C, chicken lysozyme, yeast alcohol dehydrogenase, rabbit aldolase A) were spiked into a complex
background in known concentrations in a latin square design. The experiment contained 6 mixtures,
and each mixture was analyzed in label-free LC-MS mode with 3 technical replicates (resulting
in the total of 18 runs). Each protein was represented by 7-21 peptides, and each peptide was
represented by 1-5 transition. Skyline is used for processing.

Usage
DDARawData.Skyline

Format
data.frame

Details
The raw data (input data for MSstats) is required to contain variable of ProteinName, PeptideSe-
quence, PrecursorCharge, FragmentIon, ProductCharge, IsotopeLabelType, Condition, BioRepli-
cate, Run, Intensity. The variable names should be fixed.
This is ’MSstats input’ format from Skyline used by ’MSstats_report.skyr’. The column names,
’FileName’ and ’Area’, should be changed to ’Run’ and ’Intensity’. There are two extra columns
called ’StandardType’ and ’Truncated’.’StandardType’ column can be used for normalization=’globalStandard’
in dataProcess. ’Truncated’ columns can be used to remove the truncated peaks with skylineRe-
port=TRUE in dataProcess.
If the information of one or more columns is not available for the original raw data, please retain
the column variables and type in fixed value. For example, the original raw data does not contain
12 designSampleSize

the information of PrecursorCharge and ProductCharge, we retain the column PrecursorCharge and
ProductCharge and then type in NA for all transitions in RawData.
Variable Intensity is required to be original signal without any log transformation and can be speci-
fied as the peak of height or the peak of area under curve.

Value
data.frame with the required format of MSstats.

Author(s)
Meena Choi, Olga Vitek.
Maintainer: Meena Choi (<[email protected]>)

References
Meena Choi, Ching-Yun Chang, Timothy Clough, Daniel Broudy, Trevor Killeen, Brendan MacLean
and Olga Vitek. "MSstats: an R package for statistical analysis of quantitative mass spectrometry-
based proteomic experiments" Bioinformatics, 30(17):1514-1526, 2014.
Timothy Clough, Safia Thaminy, Susanne Ragg, Ruedi Aebersold, Olga Vitek. "Statistical protein
quantification and significance analysis in label-free LC-M experiments with complex designs"
BMC Bioinformatics, 13:S16, 2012.

Examples
head(DDARawData.Skyline)

designSampleSize Planning future experimental designs of Selected Reaction Monitor-

ing (SRM), Data-Dependent Acquisition (DDA or shotgun), and Data-
Independent Acquisition (DIA or SWATH-MS) experiments in sample
size calculation

Description
Calculate sample size for future experiments of a Selected Reaction Monitoring (SRM), Data-
Dependent Acquisition (DDA or shotgun), and Data-Independent Acquisition (DIA or SWATH-
MS) experiment based on intensity-based linear model. Two options of the calculation: (1) number
of biological replicates per condition, (2) power.

Usage
designSampleSize(data=data,desiredFC=desiredFC,FDR=0.05,numSample=TRUE,power=0.9)

Arguments
data ’fittedmodel’ in testing output from function groupComparison.
desiredFC the range of a desired fold change which includes the lower and upper values of
the desired fold change.
FDR a pre-specified false discovery ratio (FDR) to control the overall false positive.
Default is 0.05
designSampleSize 13

numSample minimal number of biological replicates per condition. TRUE represents you
require to calculate the sample size for this category, else you should input the
exact number of biological replicates.
power a pre-specified statistical power which defined as the probability of detecting a
true fold change. TRUE represent you require to calculate the power for this
category, else you should input the average of power you expect. Default is 0.9

Details
The function fits the model and uses variance components to calculate sample size. The underlying
model fitting with intensity-based linear model with technical MS run replication. Estimated sample
size is rounded to 0 decimal.

Value
A list of the sample size calculation results including Variable desiredFC, numSample, numPep,
numTran, FDR, and power.

Warning
It can only obtain either one of the categories of the sample size calculation (numSample, numPep,
numTran, power) at the same time.

Author(s)
Meena Choi, Ching-Yun Chang, Olga Vitek.
Maintainer: Meena Choi (<[email protected]>)

References
Meena Choi, Ching-Yun Chang, Timothy Clough, Daniel Broudy, Trevor Killeen, Brendan MacLean
and Olga Vitek. "MSstats: an R package for statistical analysis of quantitative mass spectrometry-
based proteomic experiments" Bioinformatics, 30(17):2524-2526, 2014.
Ching-Yun Chang, Paola Picotti, Ruth Huttenhain, Viola Heinzelmann-Schwarz, Marko Jovanovic,
Ruedi Aebersold, Olga Vitek. "Protein significance analysis in selected reaction monitoring (SRM)
measurements." Molecular & Cellular Proteomics, 11:M111.014662, 2012.
Timothy Clough, Safia Thaminy, Susanne Ragg, Ruedi Aebersold, Olga Vitek. "Statistical protein
quantification and significance analysis in label-free LC-M experiments with complex designs"
BMC Bioinformatics, 13:S16, 2012.

Examples
# Consider quantitative data (i.e. QuantData) from yeast study.
# A time course study with ten time points of interests and three biological replicates.

QuantData<-dataProcess(SRMRawData)
head(QuantData$ProcessedData)

## based on multiple comparisons (T1 vs T3; T1 vs T7; T1 vs T9)

comparison1<-matrix(c(-1,0,1,0,0,0,0,0,0,0),nrow=1)
comparison2<-matrix(c(-1,0,0,0,0,0,1,0,0,0),nrow=1)
comparison3<-matrix(c(-1,0,0,0,0,0,0,0,1,0),nrow=1)
comparison<-rbind(comparison1,comparison2, comparison3)
14 designSampleSizeClassification

row.names(comparison)<-c("T3-T1","T7-T1","T9-T1")

testResultMultiComparisons<-groupComparison(contrast.matrix=comparison,data=QuantData)

## Calculate sample size for future experiments:

#(1) Minimal number of biological replicates per condition

designSampleSize(data=testResultMultiComparisons$fittedmodel, numSample=TRUE,
desiredFC=c(1.25,1.75), FDR=0.05, power=0.8)

#(2) Power calculation

designSampleSize(data=testResultMultiComparisons$fittedmodel, numSample=2,
desiredFC=c(1.25,1.75), FDR=0.05, power=TRUE)

designSampleSizeClassification
Estimate the optimal size of training data for classification problem

Description
For classification problem (such as disgnosys of disease), calculate the mean predictive accuray
under different size of training data for future experiments of a Selected Reaction Monitoring
(SRM), Data-Dependent Acquisition (DDA or shotgun), and Data-Independent Acquisition (DIA
or SWATH-MS) experiment based on simulation.

Usage
designSampleSizeClassification(data, n_sample = 5, sample_incr = 20,
protein_desc = 0.2, iter = 10)

Arguments
data output from function dataProcess
n_sample number of different sample size to simulate. Default is 5
sample_incr number of samples per condition to increase at each step. Default is 20
protein_desc the fraction of proteins to reduce at each step. Proteins are ranked based on their
mean abundance across all the samples. Default is 0.2. If protein_desc = 0.0,
protein number will not be changed.
iter number of times to repeat simulation experiments. Default is 10

Details
The function fits intensity-based linear model on the input prelimiary data data and uses variance
components and mean abundance to simulate new training data with different sample size and pro-
tein number. Random forest model is fitted on simulated train data and used to predict the input
preliminary data data. The above procedure is repeated iter times. Mean predictive accuracy and
variance under different size of training data are reported.
designSampleSizeClassificationPlots 15

Value
meanPA is the mean predictive accuracy matrix under different size of training data.
varPA is variance of predictive accuracy under different size of training data.

Author(s)
Ting Huang, Meena Choi, Olga Vitek.
Maintainer: Meena Choi (<[email protected]>)

References
T. Huang et al. TBD 2018

Examples
# Consider the training set from a colorectal cancer study
# Subjects are from control group or colorectal cancer group
# 72 proteins were targeted with SRM
require(MSstatsBioData)
set.seed(1235)
data(SRM_crc_training)
QuantCRCSRM <- dataProcess(SRM_crc_training, normalization = FALSE)
# estimate the mean predictive accuray under different sizes of training data
# n_sample is the number of different sample size to simulate
# Datasets with 10 different sample size and 3 different protein numbers are simulated
result.crc.srm <- designSampleSizeClassification(data=QuantCRCSRM,
n_sample = 10,
sample_incr = 10,
protein_desc = 0.33,
iter = 50)
result.crc.srm$meanPA # mean predictive accuracy

designSampleSizeClassificationPlots
Visualization for sample size calculation in classification problem

Description
To illustrate the mean classification accuracy under different protein number and sample size. The
input is the result from function designSampleSizeClassification.

Usage
designSampleSizeClassificationPlots(data)

Arguments
data output from function designSampleSizeClassification

Details
Data in the example is based on the results of sample size calculation in classification problem from
function designSampleSizeClassification
16 designSampleSizePlots

Value
Plot for sample size estimation. x-axis : sample size, y-axis: mean predictive accuracy. Color:
different protein number.

Author(s)
Ting Huang, Meena Choi, Olga Vitek.
Maintainer: Meena Choi (<[email protected]>)

References
T. Huang et al. TBD 2018

Examples
# Consider the training set from a colorectal cancer study
# Subjects are from control group or colorectal cancer group
# 72 proteins were targeted with SRM
require(MSstatsBioData)
set.seed(1235)
data(SRM_crc_training)
QuantCRCSRM <- dataProcess(SRM_crc_training, normalization = FALSE)
# estimate the mean predictive accuray under different sizes of training data
# n_sample is the number of different sample size to simulate
# Datasets with 10 different sample size and 3 different protein numbers are simulated
result.crc.srm <- designSampleSizeClassification(data=QuantCRCSRM,
n_sample = 10,
sample_incr = 10,
protein_desc = 0.33,
iter = 50)
designSampleSizeClassificationPlots(data=result.crc.srm)

designSampleSizePlots Visualization for sample size calculation

Description
To illustrate the relationship of desired fold change and the calculated minimal number sample
size which are (1) number of biological replicates per condition, (2) number of peptides per pro-
tein, (3) number of transitions per peptide, and (4) power. The input is the result from function
(designSampleSize.

Usage
designSampleSizePlots(data=data)

Arguments
data output from function designSampleSize.

Details
Data in the example is based on the results of sample size calculation from function designSampleSize.
designSampleSizePlots 17

Value
Plot for estimated sample size with assigned variable.

Author(s)
Meena Choi, Ching-Yun Chang, Olga Vitek.
Maintainer: Meena Choi (<[email protected]>)

References
Meena Choi, Ching-Yun Chang, Timothy Clough, Daniel Broudy, Trevor Killeen, Brendan MacLean
and Olga Vitek. "MSstats: an R package for statistical analysis of quantitative mass spectrometry-
based proteomic experiments" Bioinformatics, 30(17):2524-2526, 2014.
Ching-Yun Chang, Paola Picotti, Ruth Huttenhain, Viola Heinzelmann-Schwarz, Marko Jovanovic,
Ruedi Aebersold, Olga Vitek. "Protein significance analysis in selected reaction monitoring (SRM)
measurements." Molecular & Cellular Proteomics, 11:M111.014662, 2012.
Timothy Clough, Safia Thaminy, Susanne Ragg, Ruedi Aebersold, Olga Vitek. "Statistical protein
quantification and significance analysis in label-free LC-M experiments with complex designs"
BMC Bioinformatics, 13:S16, 2012.

Examples
# Based on the results of sample size calculation from function designSampleSize,
# we generate a series of sample size plots for number of biological replicates, or peptides,
# or transitions or power plot.

QuantData<-dataProcess(SRMRawData)
head(QuantData$ProcessedData)

## based on multiple comparisons (T1 vs T3; T1 vs T7; T1 vs T9)

comparison1<-matrix(c(-1,0,1,0,0,0,0,0,0,0),nrow=1)
comparison2<-matrix(c(-1,0,0,0,0,0,1,0,0,0),nrow=1)
comparison3<-matrix(c(-1,0,0,0,0,0,0,0,1,0),nrow=1)
comparison<-rbind(comparison1,comparison2, comparison3)
row.names(comparison)<-c("T3-T1","T7-T1","T9-T1")

testResultMultiComparisons<-groupComparison(contrast.matrix=comparison,data=QuantData)

# plot the calculated sample sizes for future experiments:

# (1) Minimal number of biological replicates per condition

result.sample<-designSampleSize(data=testResultMultiComparisons$fittedmodel, numSample=TRUE,
desiredFC=c(1.25,1.75), FDR=0.05, power=0.8)
designSampleSizePlots(data=result.sample)

# (2) Power

result.power<-designSampleSize(data=testResultMultiComparisons$fittedmodel, numSample=2,
desiredFC=c(1.25,1.75), FDR=0.05, power=TRUE)
designSampleSizePlots(data=result.power)
18 DIARawData

DIARawData Example dataset from a label-free DIA, a group comparison study of

S.Pyogenes.

Description
This example dataset was obtained from a group comparison study of S. Pyogenes. Two condi-
tions, S. Pyogenes with 0% and 10% of human plasma added (denoted Strep 0% and Strep 10%),
were profiled in two replicates, in the label-free mode, with a SWATH-MS-enabled AB SCIEX
TripleTOF 5600 System. The identification and quantification of spectral peaks was assisted by a
spectral library, and was performed using OpenSWATH software (http: //proteomics.ethz.ch/openswath.html).
For reasons of space, the example dataset only contains two proteins from this study. Protein FabG
shows strong evidence of differential abundance, while protein Probable RNA helicase exp9 only
shows moderate evidence of dif- ferential abundance between conditions.

Usage
DIARawData

Format
data.frame

Details
The raw data (input data for MSstats) is required to contain variable of ProteinName, PeptideSe-
quence, PrecursorCharge, FragmentIon, ProductCharge, IsotopeLabelType, Condition, BioRepli-
cate, Run, Intensity. The variable names should be fixed.
If the information of one or more columns is not available for the original raw data, please retain
the column variables and type in fixed value. For example, the original raw data does not contain
the information of PrecursorCharge and ProductCharge, we retain the column PrecursorCharge and
ProductCharge and then type in NA for all transitions in RawData.
Variable Intensity is required to be original signal without any log transformation and can be speci-
fied as the peak of height or the peak of area under curve.

Value
data.frame with the required format of MSstats.

Author(s)
Meena Choi, Olga Vitek.
Maintainer: Meena Choi (<[email protected]>)

Examples
head(DIARawData)
DIAUmpiretoMSstatsFormat 19

DIAUmpiretoMSstatsFormat
Generate MSstats required input format for DIA-Umpire output

Description
Convert DIA-Umpire output into the required input format for MSstats.

Usage
DIAUmpiretoMSstatsFormat(raw.frag, raw.pep, raw.pro,
annotation,
useSelectedFrag = TRUE,
useSelectedPep = TRUE,
fewMeasurements="remove",
removeProtein_with1Feature = FALSE,
summaryforMultipleRows=max)

Arguments
raw.frag name of FragSummary_date.xls data, which includes feature-level data.
raw.pep name of PeptideSummary_date.xls data, which includes selected fragments in-
formation.
raw.pro name of ProteinSummary_date.xls data, which includes selected peptides infor-
mation.
annotation name of annotation data which includes Condition, BioReplicate, Run informa-
tion.
useSelectedFrag
TRUE will use the selected fragment for each peptide. ’Selected_fragments’
column is required.
useSelectedPep TRUE will use the selected peptide for each protein. ’Selected_peptides’ col-
umn is required.
fewMeasurements
’remove’(default) will remove the features that have 1 or 2 measurements across
runs.
removeProtein_with1Feature
TRUE will remove the proteins which have only 1 feature, which is the combi-
nation of peptide, precursor charge, fragment and charge. FALSE is default.
summaryforMultipleRows
max(default) or sum - when there are multiple measurements for certain feature
and certain run, use highest or sum of multiple intensities.

Value
data.frame with the required format of MSstats.

Author(s)
Meena Choi, Olga Vitek.
Maintainer: Meena Choi (<[email protected]>)
20 groupComparison

Examples
# Manual will be updated.
# Output of DIAUmpiretoMSstatsFormat function
# should have the same 10 columns as an example dataset.

head(DDARawData)

groupComparison Finding differentially abundant proteins across conditions in tar-

geted Selected Reaction Monitoring (SRM), Data-Dependent Acqui-
sition (DDA or shotgun), and Data-Independent Acquisition (DIA or
SWATH-MS) experiment

Description
Tests for significant changes in protein abundance across conditions based on a family of linear
mixed-effects models in targeted Selected Reaction Monitoring (SRM), Data-Dependent Acquisi-
tion (DDA or shotgun), and Data-Independent Acquisition (DIA or SWATH-MS) experiment. It is
applicable to multiple types of sample preparation, including label-free workflows, workflows that
use stable isotope labeled reference proteins and peptides, and workflows that use fractionation. Ex-
perimental design of case-control study (patients are not repeatedly measured) or time course study
(patients are repeatedly measured) is automatically determined based on proper statistical model.

Usage
groupComparison(contrast.matrix=contrast.matrix,
data=data)

Arguments
contrast.matrix
comparison between conditions of interests.
data name of the (output of dataProcess function) data set.

Details
• contrast.matrix : comparison 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.
Command levels(QuantData$ProcessedData$GROUP_ORIGINAL) can illustrate the actual
order of the levels of conditions.
The underlying model fitting functions are lm and lmer for the fixed effects model and mixed effects
model, respectively.
The input of this function is the quantitative data from function (dataProcess).

Value
A list of data.frame ComparisonResult is the data.frame for the result of significance analysis ;
fittedModel is the the data.frame for run-level summarized data.
groupComparison 21

Warning

When a feature is missing completely in a condition or a MS run, a warning message is sent to

the console notifying the user of the missing feature. Additional filtering or imputing process is
required before model fitting.

Author(s)

Meena Choi, Ching-Yun Chang, Olga Vitek.

Maintainer: Meena Choi (<[email protected]>)

References

Meena Choi, Ching-Yun Chang, Timothy Clough, Daniel Broudy, Trevor Killeen, Brendan MacLean
and Olga Vitek. "MSstats: an R package for statistical analysis of quantitative mass spectrometry-
based proteomic experiments" Bioinformatics, 30(17):2524-2526, 2014.
Ching-Yun Chang, Paola Picotti, Ruth Huttenhain, Viola Heinzelmann-Schwarz, Marko Jovanovic,
Ruedi Aebersold, Olga Vitek. "Protein significance analysis in selected reaction monitoring (SRM)
measurements." Molecular & Cellular Proteomics, 11:M111.014662, 2012.
Timothy Clough, Safia Thaminy, Susanne Ragg, Ruedi Aebersold, Olga Vitek. "Statistical protein
quantification and significance analysis in label-free LC-M experiments with complex designs"
BMC Bioinformatics, 13:S16, 2012.

Examples

# Consider quantitative data (i.e. QuantData) from yeast study with ten time points of interests,
# three biological replicates, and no technical replicates.
# It is a time-course experiment and we attempt to compare differential abundance
# between time 1 and 7 in a set of targeted proteins.
# In this label-based SRM experiment, MSstats uses the fitted model with expanded scope of
# Biological replication.

QuantData <- dataProcess(SRMRawData)

head(QuantData$ProcessedData)

levels(QuantData$ProcessedData$GROUP_ORIGINAL)
comparison <- matrix(c(-1,0,0,0,0,0,1,0,0,0),nrow=1)
row.names(comparison) <- "T7-T1"

# Tests for differentially abundant proteins with models:

# label-based SRM experiment with expanded scope of biological replication.

testResultOneComparison <- groupComparison(contrast.matrix=comparison, data=QuantData)

# table for result

testResultOneComparison$ComparisonResult
22 groupComparisonPlots

groupComparisonPlots Visualization for model-based analysis and summarizing differentially

abundant proteins

Description
To summarize the results of log-fold changes and adjusted p-values for differentially abundant pro-
teins, groupComparisonPlots takes testing results from function (groupComparison) as input and
automatically generate three types of figures in pdf files as output : (1) volcano plot (specify "Vol-
canoPlot" in option type) for each comparison separately; (2) heatmap (specify "Heatmap" in option
type) for multiple comparisons ; (3) comparison plot (specify "ComparisonPlot" in option type) for
multiple comparisons per protein.

Usage
groupComparisonPlots(data=data,
type=type,
sig=0.05,
FCcutoff=FALSE,
logBase.pvalue=10,
ylimUp=FALSE,
ylimDown=FALSE,
xlimUp=FALSE,
x.axis.size=10,
y.axis.size=10,
dot.size=3,
text.size=4,
legend.size=13,
ProteinName=TRUE,
colorkey=TRUE,
numProtein=100,
clustering="both",
width=10,
height=10,
which.Comparison="all",
which.Protein="all",
address="")

Arguments
data ’ComparisonResult’ in testing output from function groupComparison.
type choice of visualization. "VolcanoPlot" represents volcano plot of log fold changes
and adjusted p-values for each comparison separately. "Heatmap" represents
heatmap of adjusted p-values for multiple comparisons. "ComparisonPlot" rep-
resents comparison plot of log fold changes for multiple comparisons per pro-
tein.
sig FDR cutoff for the adjusted p-values in heatmap and volcano plot. level of sig-
nificance for comparison plot. 100(1-sig)% confidence interval will be drawn.
sig=0.05 is default.
groupComparisonPlots 23

FCcutoff for volcano plot or heatmap, whether involve fold change cutoff or not. FALSE
(default) means no fold change cutoff is applied for significance analysis. FC-
cutoff = specific value means specific fold change cutoff is applied.
logBase.pvalue for volcano plot or heatmap, (-) logarithm transformation of adjusted p-value
with base 2 or 10(default).
ylimUp for all three plots, upper limit for y-axis. FALSE (default) for volcano plot/heatmap
use maximum of -log2 (adjusted p-value) or -log10 (adjusted p-value). FALSE
(default) for comparison plot uses maximum of log-fold change + CI.
ylimDown for all three plots, lower limit for y-axis. FALSE (default) for volcano plot/heatmap
use minimum of -log2 (adjusted p-value) or -log10 (adjusted p-value). FALSE
(default) for comparison plot uses minimum of log-fold change - CI.
xlimUp for Volcano plot, the limit for x-axis. FALSE (default) for use maximum for
absolute value of log-fold change or 3 as default if maximum for absolute value
of log-fold change is less than 3.
x.axis.size size of axes labels, e.g. name of the comparisons in heatmap, and in comparison
plot. Default is 10.
y.axis.size size of axes labels, e.g. name of targeted proteins in heatmap. Default is 10.
dot.size size of dots in volcano plot and comparison plot. Default is 3.
text.size size of ProteinName label in the graph for Volcano Plot. Default is 4.
legend.size size of legend for color at the bottom of volcano plot. Default is 7.
ProteinName for volcano plot only, whether display protein names or not. TRUE (default)
means protein names, which are significant, are displayed next to the points.
FALSE means no protein names are displayed.
colorkey TRUE(default) shows colorkey.
numProtein The number of proteins which will be presented in each heatmap. Default is
100. Maximum possible number of protein for one heatmap is 180.
clustering Determines how to order proteins and comparisons. Hierarchical cluster anal-
ysis with Ward method(minimum variance) is performed. ’protein’ means that
protein dendrogram is computed and reordered based on protein means (the or-
der of row is changed). ’comparison’ means comparison dendrogram is com-
puted and reordered based on comparison means (the order of comparison is
changed). ’both’ means to reorder both protein and comparison. Default is ’pro-
tein’.
width width of the saved file. Default is 10.
height height of the saved file. Default is 10.
which.Comparison
list of comparisons to draw plots. List can be labels of comparisons or order
numbers of comparisons from levels(data$Label), such as levels(testResultMultiComparisons$Compa
Default is "all", which generates all plots for each protein.
which.Protein Protein list to draw comparison plots. List can be names of Proteins or order
numbers of Proteins from levels(testResultMultiComparisons$ComparisonResult$Protein).
Default is "all", which generates all comparison plots for each protein.
address the name of folder that will store the results. Default folder is the current work-
ing 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 "VolcanoPlot.pdf" or "Heatmap.pdf" or "ComparisonPlot.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.
24 groupComparisonPlots

Details
• Volcano plot : illustrate actual log-fold changes and adjusted p-values for each comparison
separately with all proteins. The x-axis is the log fold change. The base of logarithm transfor-
mation is the same as specified in "logTrans" from dataProcess. The y-axis is the negative
log2 or log10 adjusted p-values. The horizontal dashed line represents the FDR cutoff. The
points below the FDR cutoff line are non-significantly abundant proteins (colored in black).
The points above the FDR cutoff line are significantly abundant proteins (colored in red/blue
for up-/down-regulated). If fold change cutoff is specified (FCcutoff = specific value), the
points above the FDR cutoff line but within the FC cutoff line are non-significantly abundant
proteins (colored in black)/
• Heatmap : illustrate up-/down-regulated proteins for multiple comparisons with all proteins.
Each column represents each comparison of interest. Each row represents each protein. Color
red/blue represents proteins in that specific comparison are significantly up-regulated/down-
regulated proteins with FDR cutoff and/or FC cutoff. The color scheme shows the evidences
of significance. The darker color it is, the stronger evidence of significance it has. Color gold
represents proteins are not significantly different in abundance.
• Comparison plot : illustrate log-fold change and its variation of multiple comparisons for
single protein. X-axis is comparison of interest. Y-axis is the log fold change. The red points
are the estimated log fold change from the model. The blue error bars are the confidence
interval with 0.95 significant level for log fold change. This interval is only based on the
standard error, which is estimated from the model.
The input of this function is "ComparisonResult" in the testing results from function (groupComparison).

Value
pdf file

Author(s)
Meena Choi, Olga Vitek.
Maintainer: Meena Choi (<[email protected]>)

References
Meena Choi, Ching-Yun Chang, Timothy Clough, Daniel Broudy, Trevor Killeen, Brendan MacLean
and Olga Vitek. "MSstats: an R package for statistical analysis of quantitative mass spectrometry-
based proteomic experiments" Bioinformatics, 30(17):2524-2526, 2014.
Ching-Yun Chang, Paola Picotti, Ruth Huttenhain, Viola Heinzelmann-Schwarz, Marko Jovanovic,
Ruedi Aebersold, Olga Vitek. "Protein significance analysis in selected reaction monitoring (SRM)
measurements." Molecular & Cellular Proteomics, 11:M111.014662, 2012.
Timothy Clough, Safia Thaminy, Susanne Ragg, Ruedi Aebersold, Olga Vitek. "Statistical protein
quantification and significance analysis in label-free LC-M experiments with complex designs"
BMC Bioinformatics, 13:S16, 2012.

Examples

QuantData<-dataProcess(SRMRawData)
head(QuantData$ProcessedData)

## based on multiple comparisons (T1 vs T3; T1 vs T7; T1 vs T9)

linear_quantlim 25

comparison1<-matrix(c(-1,0,1,0,0,0,0,0,0,0),nrow=1)
comparison2<-matrix(c(-1,0,0,0,0,0,1,0,0,0),nrow=1)
comparison3<-matrix(c(-1,0,0,0,0,0,0,0,1,0),nrow=1)
comparison<-rbind(comparison1,comparison2, comparison3)
row.names(comparison)<-c("T3-T1","T7-T1","T9-T1")

testResultMultiComparisons<-groupComparison(contrast.matrix=comparison,data=QuantData)

testResultMultiComparisons$ComparisonResult

# Volcano plot with FDR cutoff = 0.05 and no FC cutoff

groupComparisonPlots(data=testResultMultiComparisons$ComparisonResult, type="VolcanoPlot",
logBase.pvalue=2, address="Ex1_")

# Volcano plot with FDR cutoff = 0.05, FC cutoff = 70, upper y-axis limit = 100,
# and no protein name displayed
# FCcutoff=70 is for demonstration purpose
groupComparisonPlots(data=testResultMultiComparisons$ComparisonResult, type="VolcanoPlot",
FCcutoff=70, logBase.pvalue=2, ylimUp=100, ProteinName=FALSE,address="Ex2_")

# Heatmap with FDR cutoff = 0.05

groupComparisonPlots(data=testResultMultiComparisons$ComparisonResult, type="Heatmap",
logBase.pvalue=2, address="Ex1_")

# Heatmap with FDR cutoff = 0.05 and FC cutoff = 70

# FCcutoff=70 is for demonstration purpose
groupComparisonPlots(data=testResultMultiComparisons$ComparisonResult, type="Heatmap",
FCcutoff=70, logBase.pvalue=2, address="Ex2_")

# Comparison Plot
groupComparisonPlots(data=testResultMultiComparisons$ComparisonResult, type="ComparisonPlot",
address="Ex1_")

# Comparison Plot
groupComparisonPlots(data=testResultMultiComparisons$ComparisonResult, type="ComparisonPlot",
ylimUp=8, ylimDown=-1, address="Ex2_")

linear_quantlim Calculation of the LOB and LOD with a linear fit

Description

This function calculates the value of the LOB (limit of blank) and LOD (limit of detection) from
the (Concentration, Intensity) spiked in data. The function also returns the values of the linear
curve fit that allows it to be plotted. At least 2 blank samples (characterized by Intensity = 0) are
required by this function which are used to calculate the background noise. The LOB is defined as
the concentration at which the value of the linear fit is equal to the 95% upper bound of the noise.
The LOD is the concentration at which the latter is equal to the 90% lower bound of the prediction
interval (5% quantile) of the linear fit. A weighted linear fit is used with weights for every unique
concentration proportional to the inverse of variance between replicates.
26 linear_quantlim

Usage
linear_quantlim(datain, alpha = 0.05, Npoints = 100, Nbootstrap = 500)

Arguments
datain Data frame that contains the input data. The input data frame has to contain
the following columns : CONCENTRATION, INTENSITY (both of which are
measurements from the spiked in experiment) and NAME which designates the
name of the assay (e.g. the name of the peptide or protein)
alpha Probability level to estimate the LOB/LOD
Npoints Number of points to use to discretize the concentration line between 0 and the
maximum spiked concentration
Nbootstrap Number of bootstrap samples to use to calculate the prediction interval of the
fit. This number has to be increased for very low alpha values or whenever very
accurate assay characterization is required.

Details
• datain : Each line of the data frame contains one measurement from the spiked-in experiment.
Multiple different INTENSITY values for the same CONCENTRATION are assumed to cor-
respond to different replicates. Blank Samples are characterized by CONCENTRATION =
0.

Value
• Data frame that contains the output of the function. It contains the following columns: i) CON-
CENTRATION: Concentration values at which the value of the fit is calculated ii) MEAN: The
value of the curve fit iii) LOW: The value of the lower bound of the 95% prediction interval
iv) UP: The value of the upper bound of the 95% prediction interval v) LOB: The value of the
LOB (one column with identical values) vi) LOD: The value of the LOD (one column with
identical values) vii) SLOPE: Value of the slope of the linear curve fit where only the spikes
above LOD are considered viii) INTERCEPT: Value of the intercept of the linear curve fit
where only the spikes above LOD are considered ix) NAME: The name of the assay (identical
to that provided in the input) x) METHOD which is always set to LINEAR when this function
is used. Each line of the data frame corresponds to a unique concentration value at which the
value of the fit and prediction interval are evaluated. More unique concentrations values than
in the input data frame are used to increase the accuracy of the LOB/D calculations.

Warning
The LOB and LOD can only be calculated when more than 2 blank samples are included. The data
should ideally be plotted using the companion function plot_quantlim to ensure that a linear fit is
suited to the data.

Author(s)
Cyril Galitzine, Olga Vitek.
Maintainer: Cyril Galitzine (<[email protected]>), Meena Choi (<[email protected]>)

References
C. Galitzine et al. "Nonlinear regression improves accuracy of characterization of multiplexed mass
spectrometric assays" Mol Cell Proteomics, doi:10.1074/mcp.RA117.000322, 2018.
MaxQtoMSstatsFormat 27

Examples

# Consider data from a spiked-in contained in an example dataset

head(SpikeInDataLinear)

## Not run:
# Call function
linear_quantlim_out <- linear_quantlim(SpikeInDataLinear)

## End(Not run)

MaxQtoMSstatsFormat Generate MSstats required input format for MaxQuant output

Description
Convert MaxQuant output into the required input format for MSstats.

Usage
MaxQtoMSstatsFormat(evidence,
annotation,
proteinGroups,
proteinID="Proteins",
useUniquePeptide=TRUE,
summaryforMultipleRows=max,
fewMeasurements="remove",
removeMpeptides=FALSE,
removeOxidationMpeptides=FALSE,
removeProtein_with1Peptide=FALSE)

Arguments
evidence name of ’evidence.txt’ data, which includes feature-level data.
annotation name of ’annotation.txt’ data which includes Raw.file, Condition, BioReplicate,
Run, IsotopeLabelType information.
proteinGroups name of ’proteinGroups.txt’ data. It needs to matching protein group ID. If
proteinGroups=NULL, use ’Proteins’ column in ’evidence.txt’.
proteinID ’Proteins’(default) or ’Leading.razor.protein’ for Protein ID.
useUniquePeptide
TRUE(default) removes peptides that are assigned for more than one proteins.
We assume to use unique peptide for each protein.
summaryforMultipleRows
max(default) or sum - when there are multiple measurements for certain feature
and certain run, use highest or sum of all.
fewMeasurements
’remove’(default) will remove the features that have 1 or 2 measurements across
runs. ’keep’ will keep all features.
28 modelBasedQCPlots

removeMpeptides
TRUE will remove the peptides including ’M’ sequence. FALSE is default.
removeOxidationMpeptides
TRUE will remove the peptides including ’oxidation (M)’ in modification. FALSE
is default.
removeProtein_with1Peptide
TRUE will remove the proteins which have only 1 peptide and charge. FALSE
is default.

Value
data.frame with the required format of MSstats.

Warning
MSstats does not support for metabolic labeling or iTRAQ experiments.

Author(s)
Meena Choi, Olga Vitek.
Maintainer: Meena Choi (<[email protected]>)

Examples
# Please check section 4.3. Suggested workflow with MaxQuant output for DDA in MSstats user manual.
# Output of MaxQtoMSstatsFormat function should have the same 10 columns as an example dataset.

head(DDARawData)

modelBasedQCPlots Visualization for model-based quality control in fitting model

Description
To check the assumption of linear model for whole plot inference, modelBasedQCPlots takes the
results after fitting models from function (groupComparison) as input and automatically generate
two types of figures in pdf files as output : (1) normal quantile-quantile plot (specify "QQPlot" in
option type) for checking normally distributed errors.; (2) residual plot (specify "ResidualPlot" in
option type).

Usage

modelBasedQCPlots(data,
type,
axis.size=10,
dot.size=3,
text.size=7,
legend.size=7,
width=10,
height=10,
which.Protein='all',
address="")
modelBasedQCPlots 29

Arguments

data output from function groupComparison.

type choice of visualization. "QQPlots" represents normal quantile-quantile plot for
each protein after fitting models. "ResidualPlots" represents a plot of residuals
versus fitted values for each protein in the dataset.
axis.size size of axes labels. Default is 10.
dot.size size of points in the graph for residual plots and QQ plots. Default is 3.
text.size size of labeling for feature names only in normal quantile-quantile plots sepa-
rately for each feature. Default is 7.
legend.size size of legend for feature names only in residual plots. Default is 7.
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 Pro-
teins from levels(testResultOneComparison$ComparisonResult$Protein). De-
fault is "all", which generates all plots for each protein.
address the name of folder that will store the results. Default folder is the current work-
ing directory. The other assigned folder has to be existed under the current
working directory. If type="residualPlots" or "QQPlots", "ResidualPlots.pdf"
or "QQPlots.plf" will be generated. 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.

Details

Results based on statistical models for whole plot level inference are accurate as long as the assump-
tions of the model are met. The model assumes that the measurement errors are normally distributed
with mean 0 and constant variance. The assumption of a constant variance can be checked by ex-
amining the residuals from the model.

• QQPlots : a normal quantile-quantile plot for each protein is generated in order to check
whether the errors are well approximated by a normal distribution. If points fall approximately
along a straight line, then the assumption is appropriate for that protein. Only large deviations
from the line are problematic.
• ResidualPlots : The plots of residuals against predicted(fitted) values. If it shows a random
scatter, then the assumption is appropriate.

The input of this function is the result from function (groupComparison).

Value

pdf file

Author(s)

Meena Choi, Olga Vitek.

Maintainer: Meena Choi (<[email protected]>)
30 nonlinear_quantlim

References
Meena Choi, Ching-Yun Chang, Timothy Clough, Daniel Broudy, Trevor Killeen, Brendan MacLean
and Olga Vitek. "MSstats: an R package for statistical analysis of quantitative mass spectrometry-
based proteomic experiments" Bioinformatics, 30(17):2524-2526, 2014.
Ching-Yun Chang, Paola Picotti, Ruth Huttenhain, Viola Heinzelmann-Schwarz, Marko Jovanovic,
Ruedi Aebersold, Olga Vitek. "Protein significance analysis in selected reaction monitoring (SRM)
measurements." Molecular & Cellular Proteomics, 11:M111.014662, 2012.
Timothy Clough, Safia Thaminy, Susanne Ragg, Ruedi Aebersold, Olga Vitek. "Statistical protein
quantification and significance analysis in label-free LC-M experiments with complex designs"
BMC Bioinformatics, 13:S16, 2012.

Examples

QuantData <- dataProcess(SRMRawData)

head(QuantData$ProcessedData)

levels(QuantData$ProcessedData$GROUP_ORIGINAL)
comparison <- matrix(c(-1,0,0,0,0,0,1,0,0,0),nrow=1)
row.names(comparison) <- "T7-T1"

# Tests for differentially abundant proteins with models:

# label-based SRM experiment with expanded scope of biological replication.

testResultOneComparison <- groupComparison(contrast.matrix=comparison, data=QuantData)

# normal quantile-quantile plots

modelBasedQCPlots(data=testResultOneComparison, type="QQPlots", address="")

# residual plots
modelBasedQCPlots(data=testResultOneComparison, type="ResidualPlots", address="")

nonlinear_quantlim Calculation of the LOB and LOD with a nonlinear fit

Description
This function calculates the value of the LOB (limit of blank) and LOD (limit of detection) from the
(Concentration, Intensity) spiked in data. This function should be used instead of the linear function
whenever a significant threshold is present at low concentrations. Such threshold is characterized
by a signal that is dominated by noise where the mean intensity is constant and independent of
concentration. The function also returns the values of the nonlinear curve fit that allows it to be
plotted. At least 2 blank samples (characterized by Intensity = 0) are required by this function which
are used to calculate the background noise. The LOB is defined as the concentration at which the
value of the nonlinear fit is equal to the 95% upper bound of the noise. The LOD is the concentration
at which the latter is equal to the 90% lower bound (5% quantile) of the prediction interval of
the nonlinear fit. A weighted nonlinear fit is used with weights for every unique concentration
proportional to the inverse of variance between replicates. The details behind the calculation of the
nonlinear fit can be found in the Reference.
nonlinear_quantlim 31

Usage

nonlinear_quantlim(datain, alpha = 0.05, Npoints = 100, Nbootstrap = 2000)

Arguments

datain Data frame that contains the input data. The input data frame has to contain
the following columns : CONCENTRATION, INTENSITY (both of which are
measurements from the spiked in experiment) and NAME which designates the
name of the assay (e.g. the name of the peptide or protein)
alpha Probability level to estimate the LOB/LOD
Npoints Number of points to use to discretize the concentration line between 0 and the
maximum spiked concentration
Nbootstrap Number of bootstrap samples to use to calculate the prediction interval of the
fit. This number has to be increased for very low alpha values or whenever very
accurate assay characterization is required.

Details

• datain : Each line of the data frame contains one measurement from the spiked-in experiment.
Multiple different INTENSITY values for the same CONCENTRATION are assumed to cor-
respond to different replicates. Blank Samples are characterized by CONCENTRATION =
0.

Value

• Data frame that contains the output of the function. It contains the following columns: i) CON-
CENTRATION: Concentration values at which the value of the fit is calculated ii) MEAN: The
value of the curve fit iii) LOW: The value of the lower bound of the 95% prediction interval
iv) UP: The value of the upper bound of the 95% prediction interval v) LOB: The value of the
LOB (one column with identical values) vi) LOD: The value of the LOD (one column with
identical values) vii) SLOPE: Value of the slope of the linear curve fit where only the spikes
above LOD are considered viii) INTERCEPT: Value of the intercept of the linear curve fit
where only the spikes above LOD are considered ix) NAME: The name of the assay (identical
to that provided in the input) x) METHOD which is always set to NONLINEAR when this
function is used. Each line of the data frame corresponds to a unique concentration value at
which the value of the fit and prediction interval are evaluated. More unique concentrations
values than in the input data frame are used to increase the accuracy of the LOB/D calcula-
tions.

Warning

The LOB and LOD can only be calculated when more than 2 blank samples are included. The data
should ideally be plotted using the companion function plot_quantlim to ensure that the fit is suited
to the data.

Author(s)

Cyril Galitzine, Olga Vitek.

Maintainer: Cyril Galitzine (<[email protected]>), Meena Choi (<[email protected]>)
32 OpenMStoMSstatsFormat

References
C. Galitzine et al. "Nonlinear regression improves accuracy of characterization of multiplexed mass
spectrometric assays" Mol Cell Proteomics, doi:10.1074/mcp.RA117.000322, 2018.

Examples

# Consider data from a spiked-in contained in an example dataset. This dataset contains
# a significant threshold at low concentrations that is not well captured by a linear fit

head(SpikeInDataNonLinear)

## Not run:
# Call function
nonlinear_quantlim_out <- nonlinear_quantlim(SpikeInDataNonLinear)

## End(Not run)

OpenMStoMSstatsFormat Generate MSstats required input format for OpenMS output

Description
Preprocess MSstats input report from OpenSWATH and convert into the required input format for
MSstats.

Usage
OpenMStoMSstatsFormat(input,
annotation=NULL,
useUniquePeptide=TRUE,
fewMeasurements="remove",
removeProtein_with1Feature=FALSE,
summaryforMultipleRows=max)

Arguments
input name of MSstats input report from OpenMS, which includes feature(peptide
ion)-level data.
annotation name of ’annotation.txt’ data which includes Condition, BioReplicate, Run. Run
should be the same as filename.
useUniquePeptide
TRUE(default) removes peptides that are assigned for more than one proteins.
We assume to use unique peptide for each protein.
fewMeasurements
’remove’(default) will remove the features that have 1 or 2 measurements across
runs.
removeProtein_with1Feature
TRUE will remove the proteins which have only 1 peptide and charge. FALSE
is default.
OpenSWATHtoMSstatsFormat 33

summaryforMultipleRows
max(default) or sum - when there are multiple measurements for certain feature
and certain run, use highest or sum of multiple intensities.

Value
data.frame with the required format of MSstats.

Author(s)
Meena Choi, Olga Vitek.
Maintainer: Meena Choi (<[email protected]>)

Examples
# Example will be ready in next version.

OpenSWATHtoMSstatsFormat
Generate MSstats required input format for OpenSWATH output

Description
Preprocess MSstats input report from OpenSWATH and convert into the required input format for
MSstats.

Usage
OpenSWATHtoMSstatsFormat(input,
annotation = NULL,
filter_with_mscore = TRUE,
mscore_cutoff = 0.01,
useUniquePeptide = TRUE,
fewMeasurements="remove",
removeProtein_with1Feature = FALSE,
summaryforMultipleRows=max)

Arguments
input name of MSstats input report from OpenSWATH, which includes feature-level
data.
annotation name of ’annotation.txt’ data which includes Condition, BioReplicate, Run. Run
should be the same as filename.
filter_with_mscore
TRUE(default) will filter out the features that have greater than mscore_cutoff
in m_score column. Those features will be removed.
mscore_cutoff Cutoff for m_score. 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.
34 PDtoMSstatsFormat

fewMeasurements
’remove’ (default) will remove the features that have 1 or 2 measurements across
runs.
removeProtein_with1Feature
TRUE will remove the proteins which have only 1 peptide and charge. FALSE
is default.
summaryforMultipleRows
max(default) or sum - when there are multiple measurements for certain feature
and certain run, use highest or sum of multiple intensities.

Value
data.frame with the required format of MSstats.

Author(s)
Meena Choi, Olga Vitek.
Maintainer: Meena Choi (<[email protected]>)

Examples
# Example will be ready in next version.

PDtoMSstatsFormat Generate MSstats required input format for Proteome discoverer out-
put

Description
Convert Proteome discoverer output into the required input format for MSstats.

Usage
PDtoMSstatsFormat(input,
annotation,
useNumProteinsColumn=FALSE,
useUniquePeptide=TRUE,
summaryforMultipleRows=max,
fewMeasurements="remove",
removeOxidationMpeptides=FALSE,
removeProtein_with1Peptide=FALSE,
which.quantification = 'Precursor.Area',
which.proteinid = 'Protein.Group.Accessions',
which.sequence = 'Sequence' )

Arguments
input name of Proteome discover PSM output, which is long-format. "Protein.Group.Accessions",
"#Proteins", "Sequence", "Modifications", "Charge", "Intensity", "Spectrum.File"
are required.
annotation name of ’annotation.txt’ or ’annotation.csv’ data which includes Condition, BioRepli-
cate, Run information. ’Run’ will be matched with ’Spectrum.File’.
PDtoMSstatsFormat 35

useNumProteinsColumn
TRUE removes peptides which have more than 1 in # Proteins column of PD
output.
useUniquePeptide
TRUE(default) removes peptides that are assigned for more than one proteins.
We assume to use unique peptide for each protein.
summaryforMultipleRows
max(default) or sum - when there are multiple measurements for certain feature
and certain run, use highest or sum of multiple intensities.
fewMeasurements
’remove’(default) will remove the features that have 1 or 2 measurements across
runs.
removeOxidationMpeptides
TRUE will remove the modified peptides including ’Oxidation (M)’ in ’Modifi-
cations’ column. FALSE is default.
removeProtein_with1Peptide
TRUE will remove the proteins which have only 1 peptide and charge. FALSE
is default.
which.quantification
Use ’Precursor.Area’(default) column for quantified intensities. ’Intensity’ or
’Area’ can be used instead.
which.proteinid
Use ’Protein.Accessions’(default) column for protein name. ’Master.Protein.Accessions’
can be used instead.
which.sequence Use ’Sequence’(default) column for peptide sequence. ’Annotated.Sequence’
can be used instead.

Value

data.frame with the required format of MSstats.

Author(s)

Meena Choi, Olga Vitek.

Maintainer: Meena Choi (<[email protected]>)

Examples

# Please check section 4.5.

## Suggested workflow with Proteome Discoverer output for DDA in MSstats user manual.
# Output of PDtoMSstatsFormat function should have the same 10 columns as an example dataset.

head(DDARawData)
36 plot_quantlim

plot_quantlim Plot of the curve used to calculate LOB and LOD

Description
This function allows to plot the curve fit that is used to calculate the LOB and LOD with functions
nonlinear_quantlim() and linear_quantlim(). The function outputs for each calibration curve, two
pdf files each containg one plot. On the first, designated by *_overall.pdf, the entire concentration
range is plotted. On the second plot, designated by *_zoom.pdf„ the concentration range between
0 and xlim_plot (if specified in the argument of the function) is plotted. When no xlim_plot value
is specified, the region close to LOB and LOD is automatically plotted.

Usage
plot_quantlim(spikeindata, quantlim_out, alpha, dir_output, xlim_plot)

Arguments
spikeindata Data frame that contains the experimental spiked in data. This data frame should
be identical to that used as input by function functions nonlinear_quantlim()
or linear_quantlim(). The data frame has to contain the following columns :
CONCENTRATION, INTENSITY (both of which are measurements from the
spiked in experiment) and NAME which designates the name of the assay (e.g.
the name of the peptide or protein)
quantlim_out Data frame that was output by functions nonlinear_quantlim() or linear_quantlim().
It has to contain at least the following columns: i) CONCENTRATION: Con-
centration values at which the value of the fit is calculated ii) MEAN: The value
of the curve fit iii) LOW: The value of the lower bound of the 95% prediction
interval iv) UP: The value of the upper bound of the 95% prediction interval v)
LOB: The value of the LOB (one column with identical values) vi) LOD: The
value of the LOD (one column with identical values) vii) NAME: The name
of the assay (identical to that provided in the input) viii) METHOD which is
LINEAR or NONLINEAR
alpha Probability level to estimate the LOB/LOD
dir_output String containg the path of the directly where the pdf files of the plots should be
output.
xlim_plot Optional argument containing the maximum xaxis value of the zoom plot. When
no value is specified, a suitable value close to LOD is automatically chosen.

Details
• spikeindata : Each line of the data frame contains one measurement from the spiked-in exper-
iment. Multiple different INTENSITY values for the same CONCENTRATION are assumed
to correspond to different replicates. Blank Samples are characterized by CONCENTRATION
= 0.

Value
• Data frame where each line corresponds to a unique concentration value at which the value
of the fit and prediction interval are evaluated. More unique concentrations values than in the
input data frame are used to increase the accuracy of the LOB/D calculations.
ProgenesistoMSstatsFormat 37

Warning

This plotting function should ideally be used every time nonlinear_quantlim() or linear_quantlim()
are called to visually ensure that the fits and data are accurate.

Author(s)

Cyril Galitzine, Olga Vitek.

Maintainer: Cyril Galitzine (<[email protected]>), Meena Choi (<[email protected]>)

References

C. Galitzine et al. "Nonlinear regression improves accuracy of characterization of multiplexed mass

spectrometric assays" Mol Cell Proteomics, doi:10.1074/mcp.RA117.000322, 2018.

Examples

# Consider data from a spiked-in contained in an example dataset. This dataset contains
# a significant threshold at low concentrations that is not well captured by a linear fit.

head(SpikeInDataNonLinear)

## Not run:
#Call function
nonlinear_quantlim_out <- nonlinear_quantlim(SpikeInDataNonLinear, alpha = 0.05)

plot_quantlim(spikeindata = SpikeInDataLinear, quantlim_out = nonlinear_quantlim_out,

dir_output = getwd(), alpha = 0.05)

## End(Not run)

ProgenesistoMSstatsFormat
Generate MSstats required input format for Progenesis output

Description

Convert Progenesis output into the required input format for MSstats.

Usage

ProgenesistoMSstatsFormat(input,
annotation,
useUniquePeptide=TRUE,
summaryforMultipleRows=max,
fewMeasurements="remove",
removeOxidationMpeptides=FALSE,
removeProtein_with1Peptide=FALSE)
38 quantification

Arguments
input name of Progenesis output, which is wide-format. ’Accession’, ’Sequence’,
’Modification’, ’Charge’ and one column for each run are required.
annotation name of ’annotation.txt’ or ’annotation.csv’ data which includes Condition, BioRepli-
cate, Run information. It will be matched with the column name of input for MS
runs.
useUniquePeptide
TRUE(default) removes peptides that are assigned for more than one proteins.
We assume to use unique peptide for each protein.
summaryforMultipleRows
max(default) or sum - when there are multiple measurements for certain feature
and certain run, use highest or sum of multiple intensities.
fewMeasurements
’remove’(default) will remove the features that have 1 or 2 measurements across
runs.
removeOxidationMpeptides
TRUE will remove the modified peptides including ’Oxidation (M)’ sequence.
FALSE is default.
removeProtein_with1Peptide
TRUE will remove the proteins which have only 1 peptide and charge. FALSE
is default.

Value
data.frame with the required format of MSstats.

Author(s)
Meena Choi, Olga Vitek.
Maintainer: Meena Choi (<[email protected]>)

Examples
# Please check section 4.4.
# Suggested workflow with Progenesis output for DDA in MSstats user manual.
# Output of ProgenesistoMSstatsFormat function
# should have the same 10 columns as an example dataset.

head(DDARawData)

quantification Protein sample quantification or group quantification

Description
Model-based quantification for each condition or for each biological samples per protein in a tar-
geted Selected Reaction Monitoring (SRM), Data-Dependent Acquisition (DDA or shotgun), and
Data-Independent Acquisition (DIA or SWATH-MS) experiment. Quantification takes the pro-
cessed data set by dataProcess as input and automatically generate the quantification results
(data.frame) with long or matrix format.
quantification 39

Usage
quantification(data, type="Sample", format="matrix")

Arguments
data name of the (processed) data set.
type choice of quantification. "Sample" or "Group" for protein sample quantification
or group quantification.
format choice of returned format. "long" for long format which has the columns named
Protein, Condition, LonIntensities (and BioReplicate if it is subject quantifica-
tion), NumFeature for number of transitions for a protein, and NumPeaks for
number of observed peak intensities for a protein. "matrix" for data matrix
format which has the rows for Protein and the columns, which are Groups(or
Conditions) for group quantification or the combinations of BioReplicate and
Condition (labeled by "BioReplicate"_"Condition") for sample quantification.
Default is "matrix"

Details
• Sample quantification : individual biological sample quantification for each protein. The label
of each biological sample is a combination of the corresponding group and the sample ID. If
there are no technical replicates or experimental replicates per sample, sample quantification
is the same as run summarization from dataProcess. If there are technical replicates or exper-
imental replicates, sample quantification is median among run quantification corresponding
MS runs.
• Group quantification : quantification for individual group or individual condition per protein.
It is median among sample quantification.
• The quantification for endogenous samples is based on run summarization from subplot model,
with TMP robust estimation.
The input of this function is the quantitative data from function (dataProcess).

Value
data.frame as described in details.

Author(s)
Meena Choi, Olga Vitek.
Maintainer: Meena Choi (<[email protected]>)

References
Meena Choi, Ching-Yun Chang, Timothy Clough, Daniel Broudy, Trevor Killeen, Brendan MacLean
and Olga Vitek. "MSstats: an R package for statistical analysis of quantitative mass spectrometry-
based proteomic experiments" Bioinformatics, 30(17):2524-2526, 2014.
Ching-Yun Chang, Paola Picotti, Ruth Huttenhain, Viola Heinzelmann-Schwarz, Marko Jovanovic,
Ruedi Aebersold, Olga Vitek. "Protein significance analysis in selected reaction monitoring (SRM)
measurements." Molecular & Cellular Proteomics, 11:M111.014662, 2012.
Timothy Clough, Safia Thaminy, Susanne Ragg, Ruedi Aebersold, Olga Vitek. "Statistical protein
quantification and significance analysis in label-free LC-M experiments with complex designs"
BMC Bioinformatics, 13:S16, 2012.
40 SkylinetoMSstatsFormat

Examples
# Consider quantitative data (i.e. QuantData) from a yeast study with ten time points of
# interests, three biological replicates, and no technical replicates which is
# a time-course experiment.
# Sample quantification shows model-based estimation of protein abundance in each biological
# replicate within each time point.
# Group quantification shows model-based estimation of protein abundance in each time point.

QuantData<-dataProcess(SRMRawData)
head(QuantData$ProcessedData)

# Sample quantification

sampleQuant<-quantification(QuantData)
head(sampleQuant)

# Group quantification

groupQuant<-quantification(QuantData, type="Group")
head(groupQuant)

SkylinetoMSstatsFormat
Generate MSstats required input format for Skyline output

Description
Preprocess MSstats input report from Skyline and convert into the required input format for MSstats.

Usage
SkylinetoMSstatsFormat(input,
annotation = NULL,
removeiRT = TRUE,
filter_with_Qvalue = TRUE,
qvalue_cutoff = 0.01,
useUniquePeptide = TRUE,
fewMeasurements="remove",
removeOxidationMpeptides = FALSE,
removeProtein_with1Feature = FALSE)

Arguments
input name of MSstats input report from Skyline, which includes feature-level data.
annotation name of ’annotation.txt’ data which includes Condition, BioReplicate, Run. If
annotation is already complete in Skyline, use annotation=NULL (default). It
will use the annotation information from input.
removeiRT TRUE(default) will remove the proteins or peptides which are labeld ’iRT’ in
’StandardType’ column. FALSE will keep them.
SpectronauttoMSstatsFormat 41

filter_with_Qvalue
TRUE(default) will filter out the intensities that have greater than qvalue_cutoff
in DetectionQValue column. Those intensities will be replaced with zero and
will be considered as censored missing values for imputation purpose.
qvalue_cutoff Cutoff for DetectionQValue. 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.
fewMeasurements
’remove’ (default) will remove the features that have 1 or 2 measurements across
runs.
removeOxidationMpeptides
TRUE will remove the peptides including ’oxidation (M)’ in modification. FALSE
is default.
removeProtein_with1Feature
TRUE will remove the proteins which have only 1 peptide and charge. FALSE
is default.

Value
data.frame with the required format of MSstats.

Author(s)
Meena Choi, Olga Vitek.
Maintainer: Meena Choi (<[email protected]>)

Examples
# Please check section 4.2. Suggested workflow with Skyline output for DDA in MSstats user manual.
# Output of SkylinetoMSstatsFormat function should have the same 10 columns as an example dataset.

head(DDARawData)

SpectronauttoMSstatsFormat
Generate MSstats required input format for Spectronaut output

Description
Convert Spectronaut output into the required input format for MSstats.

Usage
SpectronauttoMSstatsFormat(input,
annotation = NULL,
intensity = 'PeakArea',
filter_with_Qvalue = TRUE,
qvalue_cutoff = 0.01,
useUniquePeptide = TRUE,
fewMeasurements="remove",
removeProtein_with1Feature = FALSE,
summaryforMultipleRows=max)
42 SpectronauttoMSstatsFormat

Arguments

input name of Spectronaut output, which is long-format. ProteinName, PeptideSe-

quence, PrecursorCharge, FragmentIon, ProductCharge, IsotopeLabelType, Con-
dition, BioReplicate, Run, Intensity, F.ExcludedFromQuantification are required.
Rows with F.ExcludedFromQuantification=True will be removed.
annotation name of ’annotation.txt’ data which includes Condition, BioReplicate, Run. If
annotation is already complete in Spectronaut, use annotation=NULL (default).
It will use the annotation information from input.
intensity ’PeakArea’(default) uses not normalized peak area. ’NormalizedPeakArea’ uses
peak area normalized by Spectronaut.
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 zero 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.
fewMeasurements
’remove’ (default) will remove the features that have 1 or 2 measurements across
runs.
removeProtein_with1Feature
TRUE will remove the proteins which have only 1 feature, which is the combi-
nation of peptide, precursor charge, fragment and charge. FALSE is default.
summaryforMultipleRows
max(default) or sum - when there are multiple measurements for certain feature
and certain run, use highest or sum of multiple intensities.

Value

data.frame with the required format of MSstats.

Author(s)

Meena Choi, Olga Vitek.

Maintainer: Meena Choi (<[email protected]>)

Examples
# Please check section 5.2.
# Suggested workflow with Spectronaut output for DIA in MSstats user manual.
# Output of SpectronauttoMSstatsFormat function
# should have the same 10 columns as an example dataset.

head(DDARawData)
SpikeInDataLinear 43

SpikeInDataLinear Example dataset from an MRM spike-in experiment with a linear be-
havior

Description

This dataset is part of the CPTAC 7, study 3 (Addona et al., 2009). It corresponds to the spike-
in data for peptide AGLCQTFVYGGCR at site 86. This particular data was chosen because it
illustrates well a linear response for a spiked in experiment. The data is composed of 4 replicates at
10 different concentrations (including a blank sample with concentration 0).

Usage

SpikeInDataLinear

Format

data.frame

Details

The intensity reported is the sum of the intensity of all the different fragments of the peptide. Only
the peptide being spiked (light peptide) is contained in the example data set. The intensity was
normalized using the corresponding heavy peptide in log space such that intensity of the heavy
remains constant for all concentrations and all replicates. The intensity was rescaled following
the method described in Addona et al., 2009. The concentration and Intensity are both in units of
fmol/uL.

Value

data.frame as described in details.

Author(s)

Cyril Galitzine, Olga Vitek.

Maintainer: Cyril Galitzine (<[email protected]>), Meena Choi (<[email protected]>)

References

T.A. Addonna et al. "Multi-site assessment of the precision and reproducibility of multiple reaction
monitoring-based measurements of proteins in plasma." Nat Biotechnol. 2009 Jul;27(7):633-41

Examples
head(SpikeInDataLinear)
44 SpikeInDataNonLinear

SpikeInDataNonLinear Example dataset from an MRM spike-in experiment with a nonlinear

behavior

Description

This dataset is part of the CPTAC 7, study 3 (Addona et al., 2009). It corresponds to the spike-in data
for peptide ESDTSYVSLK at site 19. This particular data was chosen because of the concentration
threshold that is present at low concentrations that warrant the use of a nonlinear method. The data is
composed of 4 replicates at 10 different concentrations (including a blank sample with concentration
0).

Usage

SpikeInDataNonLinear

Format

data.frame

Details

The intensity reported is the sum of the intensity of all the different fragments of the peptide. Only
the peptide being spiked (light peptide) is contained in the example data set. The intensity was
normalized using the corresponding heavy peptide in log space such that intensity of the heavy
remains constant for all concentrations and all replicates. The intensity was rescaled following
the method described in Addona et al., 2009. The concentration and Intensity are both in units of
fmol/uL.

Value

data.frame as described in details.

Author(s)

Cyril Galitzine, Olga Vitek.

Maintainer: Cyril Galitzine (<[email protected]>), Meena Choi (<[email protected]>)

References

T.A. Addonna et al. "Multi-site assessment of the precision and reproducibility of multiple reaction
monitoring-based measurements of proteins in plasma." Nat Biotechnol. 2009 Jul;27(7):633-41

Examples
head(SpikeInDataNonLinear)
SRMRawData 45

SRMRawData Example dataset from a SRM experiment with stable isotope labeled
reference of a time course yeast study

Description
This is a partial data set obtained from a published study (Picotti, et. al, 2009). The experiment
targeted 45 proteins in the glycolysis/gluconeogenesis/TCA cycle/glyoxylate cycle network, which
spans the range of protein abundance from less than 128 to 10E6 copies per cell. Three biological
replicates were analyzed at ten time points (T1-T10), while yeasts transited through exponential
growth in a glucose-rich medium (T1-T4), diauxic shift (T5-T6), post-diauxic phase (T7-T9), and
stationary phase (T10). Prior to trypsinization, the samples were mixed with an equal amount of
proteins from the same N15-labeled yeast sample, which was used as a reference. Each sample
was profiled in a single mass spectrometry run, where each protein was represented by up to two
peptides and each peptide by up to three transitions. The goal of this study is to detect significantly
change in protein abundance across time points. Transcriptional activity under the same experimen-
tal conditions has been previously investigated by (DeRisi et. al., 1997). Genes coding for 29 of
the proteins are differentially expressed between conditions similar to those represented by T7 and
T1 and could be treated as external sources to validate the proteomics analysis. In this exampled
data set, two of the targeted proteins are selected and validated with gene expression study: Pro-
tein IDHC (gene name IDP2) is differentially expressed in time point 1 and time point 7, whereas,
Protein PMG2 (gene name GPM2) is not. The protein names are based on Swiss Prot Name.

Usage
SRMRawData

Format
data.frame

Details
The raw data (input data for MSstats) is required to contain variable of ProteinName, PeptideSe-
quence, PrecursorCharge, FragmentIon, ProductCharge, IsotopeLabelType, Condition, BioRepli-
cate, Run, Intensity. The variable names should be fixed.
If the information of one or more columns is not available for the original raw data, please retain
the column variables and type in fixed value. For example, the original raw data does not contain
the information of ProductCharge, we retain the column ProductCharge and type in NA for all
transitions in RawData.
The column BioReplicate should label with unique patient ID (i.e., same patients should label with
the same ID).
Variable Intensity is required to be original signal without any log transformation and can be speci-
fied as the peak of height or the peak of area under curve.

Value
data.frame with the required format of MSstats.
46 SRMRawData

Author(s)
Meena Choi, Olga Vitek.
Maintainer: Meena Choi (<[email protected]>)

References
Ching-Yun Chang, Paola Picotti, Ruth Huttenhain, Viola Heinzelmann-Schwarz, Marko Jovanovic,
Ruedi Aebersold, Olga Vitek. Protein significance analysis in selected reaction monitoring (SRM)
measurements. Molecular & Cellular Proteomics, 11:M111.014662, 2012.

Examples
head(SRMRawData)
Index

∗Topic MSstats
MSstats-package, 2

dataProcess, 3, 3, 7, 9, 11, 14, 20, 24, 38, 39

dataProcessPlots, 3, 7
DDARawData, 3, 10
DDARawData.Skyline, 11
designSampleSize, 3, 12, 16
designSampleSizeClassification, 14, 15
designSampleSizeClassificationPlots,
15
designSampleSizePlots, 3, 16
DIARawData, 3, 18
DIAUmpiretoMSstatsFormat, 3, 19

groupComparison, 3, 20, 22, 24, 28, 29

groupComparisonPlots, 3, 22

linear_quantlim, 25
lm, 20
lmer, 20

MaxQtoMSstatsFormat, 3, 27
modelBasedQCPlots, 3, 28
MSstats (MSstats-package), 2
MSstats-package, 2

nonlinear_quantlim, 30

OpenMStoMSstatsFormat, 3, 32
OpenSWATHtoMSstatsFormat, 3, 33

PDtoMSstatsFormat, 3, 34
plot_quantlim, 36
ProgenesistoMSstatsFormat, 3, 37

quantification, 3, 38

SkylinetoMSstatsFormat, 3, 40
SpectronauttoMSstatsFormat, 3, 41
SpikeInDataLinear, 43
SpikeInDataNonLinear, 44
SRMRawData, 3, 5, 45

47