# Purpose of MSstatsConvert

The MSstatsConvert package is a member of the MSstatst family of packages, MSstats and MSstatsTMT. It creates an abstraction for the steps in mass spectrometry (MS) data analysis that are required before a dataset can be used for statistical modeling. In short, the package is responsible for converting output from signal processing tools such as OpenMS or MaxQuant into a format suitable for statistical analysis. This includes:

• PSM- and protein-level filtering,
• managing shared peptides,
• removing decoy, iRT, and other irrelavant sequences,
• removing features or proteins with a low number of measurements,
• aggregating duplicated measurements,
• handling fractionation by removing overlapped features,

MSstatsConvert allows for transforming any MS quantification result into a format required by MSstats and MSstatsTMT packages. Additionally, it provides built-in cleaning functions for outputs of DIAUmpire, MaxQuant, OpenMS, OpenSWATH, Progenesis, ProteomeDiscoverer, Skyline, Spectromine, and Spectronaut. These functions serve as a base for converter functions (called *toMSstatsFormat or *toMSstatsTMTFormat) provided by the MSstats and MSstatsTMT packages.

# MSstats data format

MSstats family packages works with label-free, SRM and TMT datasets. The following column are required.

• ProteinName: column that indicates a protein ID. If the analysis is to be made at the peptide-level, the column should store peptide IDs. Summarization performed by MSstats and MSstatsTMT packages is done separately for each ID in this column,
• PeptideSequence, PrecursorCharge, FragmentIon and ProductCharge: these four columns define a spectral feature (transition in SRM case). If information for any of the columns is not available, it should be set to a constant value (for example NA),
• IsotopeLabelType: column that indicates whether the measurement is based on an endogenous peptide (indicated by value “L” or “light”) or reference peptide (indicated by value “H” or “heavy”),
• Run: column that stores IDs of mass spectrometry runs. If annotation describing biological conditions and replicates is provided via a separate table, the run IDs should match Run IDs in the annotation,
• Condition: column that stores labels for biological conditions (groups of interest). For time-course experiments, this column will represent time points. If the design experiment includes both time points and distinct biological subjects, these labels should be a combination of subject and time point,
• BioReplicate: this column should contain a unique identifier for each biological replicate in the experiment. For example, in a clinical proteomic investigation this should be a unique patient ID. Patients from distinct groups (indicated by the Condition column) should have distinct IDs,
• Intensity: column that stores untransformed (in particular, no log transformation) measurements of feature abundance in a given Run (and Channel in TMT case). They can be peak heights, peak areas under the curve, or other quantitative representations of feature abundance,
• for TMT datasets, a Channel column is required. Similarly to the Run column, values in this column must correspond to values in the annotation file, if provided separately.

Additionally, if the experiment involves fractionation, Fraction column can be added to store fraction labels.

# Logging

MSstatsConvert allows for flexible logging based on the log4r package. Information about preprocessing steps can be written to a file, to a console, to both or to neither. The MSstatsLogsSettings function helps manage log settings. The user can pass a path to an existing file to the log_file_path parameter. Combined with setting append = TRUE, this allows writing all information related to a specific data analysis to a single file. If a user does not specify a file, a new file will be created automatically with a name starting with “MSstats_log”, followed by a timestamp.

library(MSstatsConvert)
# default - creates a new file
MSstatsLogsSettings(use_log_file = TRUE, append = FALSE)

# default - creates a new file
MSstatsLogsSettings(use_log_file = TRUE, append = TRUE,
log_file_path = "log_file.log")

# switches logging off
MSstatsLogsSettings(use_log_file = FALSE, append = FALSE)

# switches off logs and messages
MSstatsLogsSettings(use_log_file = FALSE, verbose = FALSE) 

Additionally, session info generated by the utils::sessionInfo() function can be saved to file with the MSstatsSaveSessionInfo function.

MSstatsSaveSessionInfo()

By default, the output file name will start with “MSstats_session_info” and end with a current timestamp.

# Importing and cleaning data

MS data processing by MSstatsConvert starts with importing and cleaning data. The MSstatsImport function produces a wrapper for possibly multiple files that may describe a single dataset. For example, MaxQuant output consists of two files, while OpenMS outputs just a single file.

maxquant_evidence = read.csv(system.file("tinytest/raw_data/MaxQuant/mq_ev.csv",
package = "MSstatsConvert"))
package = "MSstatsConvert"))
maxquant_imported = MSstatsImport(list(evidence = maxquant_evidence,
protein_groups = maxquant_proteins),
type = "MSstats", tool = "MaxQuant")
is(maxquant_imported)
#> [1] "MSstatsMaxQuantFiles" "MSstatsInputFiles"

"tinytest/raw_data/OpenMSTMT/openmstmt_input.csv",
package = "MSstatsConvert"
))
openms_imported = MSstatsImport(list(input = openms_input),
"MSstatsTMT", "OpenMS")
is(openms_imported)
#> [1] "MSstatsOpenMSFiles" "MSstatsInputFiles"

The getInputFile method allows user to retrieve the files:

getInputFile(maxquant_imported, "evidence")[1:5, 1:5]
#>       Sequence Length Modifications Modifiedsequence OxidationMProbabilities
#> 1: AEAPAAAPAAK     11    Unmodified    _AEAPAAAPAAK_
#> 2: AEAPAAAPAAK     11    Unmodified    _AEAPAAAPAAK_
#> 3: AEAPAAAPAAK     11    Unmodified    _AEAPAAAPAAK_
#> 4: AEAPAAAPAAK     11    Unmodified    _AEAPAAAPAAK_
#> 5: AEAPAAAPAAK     11    Unmodified    _AEAPAAAPAAK_

As a next step of the analysis, input files are combined into a single data.table with standardized column names by the MSstatsClean function. It is a generic function with built-in support for outputs of tools listed in the “Purpose of the MSstatsConvert package” section. The type parameter is equal to either MSstats or MSstatsTMT and indicates if the data comes from a labelled TMT experiment.

For some datasets, MSstatsClean may require additional parameters described in the respective help files. For our example datasets, the following calls merge input files into a single table.

maxquant_cleaned = MSstatsClean(maxquant_imported, protein_id_col = "Proteins")
#>    ProteinName PeptideSequence Modifications PrecursorCharge
#> 1:      P06959     AEAPAAAPAAK    Unmodified               2
#> 2:      P06959     AEAPAAAPAAK    Unmodified               2
#> 3:      P06959     AEAPAAAPAAK    Unmodified               2
#> 4:      P06959     AEAPAAAPAAK    Unmodified               2
#> 5:      P06959     AEAPAAAPAAK    Unmodified               2
#> 6:      P06959     AEAPAAAPAAK    Unmodified               2
#>                                            Run Intensity   Score
#> 1: 121219_S_CCES_01_01_LysC_Try_1to10_Mixt_1_1   4023100  76.332
#> 2: 121219_S_CCES_01_02_LysC_Try_1to10_Mixt_1_2   5132500  83.081
#> 3: 121219_S_CCES_01_03_LysC_Try_1to10_Mixt_1_3   2761600 104.430
#> 4: 121219_S_CCES_01_05_LysC_Try_1to10_Mixt_2_2   4091800  94.465
#> 5: 121219_S_CCES_01_06_LysC_Try_1to10_Mixt_2_3   4727000  88.596
#> 6: 121219_S_CCES_01_08_LysC_Try_1to10_Mixt_3_2   2258400  90.050

openms_cleaned = MSstatsClean(openms_imported)
#>             ProteinName                               PeptideSequence
#> 1: sp|Q60854|SPB6_MOUSE .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR
#> 2: sp|Q60854|SPB6_MOUSE .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR
#> 3: sp|Q60854|SPB6_MOUSE .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR
#> 4: sp|Q60854|SPB6_MOUSE .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR
#> 5: sp|Q60854|SPB6_MOUSE .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR
#> 6: sp|Q60854|SPB6_MOUSE .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR
#>    PrecursorCharge
#> 1:               3
#> 2:               3
#> 3:               3
#> 4:               3
#> 5:               3
#> 6:               3
#>                                                                 PSM Condition
#> 1: .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR_3_4359.56536443198   Long_HF
#> 2: .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR_3_6190.04195694402   Long_HF
#> 3: .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR_3_4359.56536443198   Long_HF
#> 4: .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR_3_6190.04195694402   Long_HF
#> 5: .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR_3_6190.04195694402   Long_HF
#> 6: .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR_3_4359.56536443198   Long_HF
#>    BioReplicate   Run Channel Intensity Mixture TechRepMixture Fraction
#> 1:           21 3_3_3       1        NA       3            3_3        3
#> 2:           21 3_3_3       1        NA       3            3_3        3
#> 3:           24 3_3_3       4        NA       3            3_3        3
#> 4:           24 3_3_3       4        NA       3            3_3        3
#> 5:           26 3_3_3       6        NA       3            3_3        3
#> 6:           26 3_3_3       6  1820.072       3            3_3        3

If a user wants to use MSstatsConvert package with data in a format that is not currently supported, it is enough to first re-format the data into a data.table with column ProteinName, PeptideSequence, PrecursorCharge, FragmentIon, ProductCharge (with the latter two possibly equal to NA), Run and IsotopeLabelType (in case of non-TMT data) or Channel (in case of TMT data). Moreover, the dataset may include any column that will be used for filtering the dataset (for example a column that stores q-values). In our example, such additional columns are “Modifications” and “Score” from MaxQuant files.

Annotation columns should be called Condition and BioReplicate. For TMT data, Mixture, TechRepMixture columns may be added. Fractionation should be indicated by a Fraction column.

# Preprocessing

The goal of MSstatsPreprocess function is to transform cleaned MS data into a format ready for statistical analysis with MSstats or MSstatsTMT packages. This function accepts several parameters, and each corresponds to a preprocessing step.

• input parameter is the dataset for preprocessing,
• annotation is a description of biological conditions and replicates associated with MS runs (and channels in TMT case). If annotation is already included in the input, it should be equal to NULL. The annotation should be created by the MSstatsMakeAnnotation function,
• feature_columns is a vector of column names that will denote features,
• remove_shared_peptides is a logical parameter - if TRUE, shared peptides will be removed from the analysis. Currently, MSstats assumes that only unique peptides are used, and presence of shared peptides may cause issues,
• remove_single_feature_proteins is a logical parameter that indicates if proteins that only have a single feature should be removed from the analysis (TRUE),
• feature_cleaning is a list, that currently consists of two named elements: remove_features_with_few_measurements should be equal to TRUE or FALSE. In the first case, feature that have less than three measurements across runs (or channels in a run for TMT data) will be removed. FALSE means that only features with no non-missing measurements will be removed. The summarize_multiple_psms element should be a function that will be used to aggregate multiple feature measurements within a single MS run,
• aggregate_isotopic is a logical parameter - TRUE means that isotopic peaks will be aggregated (currently only used for Skyline input),
• columns_to_fill is an optional named list with names corresponding to columns and values correponding to values that will be used for these columns. For example, if the dataset is missing information about ProductCharge, such a column can be added by passing list(ProductCharge = NA) to this parameter,
• score_filtering, exact_filtering and pattern_filtering parameters are optional parameters that can be used to perform data filtering. An example is given below.
maxquant_annotation = read.csv(system.file(
"tinytest/raw_data/MaxQuant/annotation.csv",
package = "MSstatsConvert"
))
maxquant_annotation = MSstatsMakeAnnotation(maxquant_cleaned,
maxquant_annotation,
Run = "Rawfile")
m_filter = list(col_name = "PeptideSequence",
pattern = "M",
filter = TRUE,
drop_column = FALSE)

oxidation_filter = list(col_name = "Modifications",
pattern = "Oxidation",
filter = TRUE,
drop_column = TRUE)

feature_columns = c("PeptideSequence", "PrecursorCharge")
maxquant_processed = MSstatsPreprocess(
maxquant_cleaned,
maxquant_annotation,
feature_columns,
remove_shared_peptides = TRUE,
remove_single_feature_proteins = FALSE,
pattern_filtering = list(oxidation = oxidation_filter,
m = m_filter),
feature_cleaning = list(remove_features_with_few_measurements = TRUE,
summarize_multiple_psms = max),
columns_to_fill = list("FragmentIon" = NA,
"ProductCharge" = NA,
"IsotopeLabelType" = "L"))
#>                                            Run PeptideSequence PrecursorCharge
#> 1: 121219_S_CCES_01_01_LysC_Try_1to10_Mixt_1_1     AEAPAAAPAAK               2
#> 2: 121219_S_CCES_01_02_LysC_Try_1to10_Mixt_1_2     AEAPAAAPAAK               2
#> 3: 121219_S_CCES_01_03_LysC_Try_1to10_Mixt_1_3     AEAPAAAPAAK               2
#> 4: 121219_S_CCES_01_05_LysC_Try_1to10_Mixt_2_2     AEAPAAAPAAK               2
#> 5: 121219_S_CCES_01_06_LysC_Try_1to10_Mixt_2_3     AEAPAAAPAAK               2
#> 6: 121219_S_CCES_01_08_LysC_Try_1to10_Mixt_3_2     AEAPAAAPAAK               2
#>    Intensity ProteinName Condition BioReplicate Experiment IsotopeLabelType
#> 1:   4023100      P06959         1            1        1_1                L
#> 2:   5132500      P06959         1            1        1_2                L
#> 3:   2761600      P06959         1            1        1_3                L
#> 4:   4091800      P06959         2            2        2_2                L
#> 5:   4727000      P06959         2            2        2_3                L
#> 6:   2258400      P06959         3            3        3_2                L
#>    FragmentIon ProductCharge
#> 1:          NA            NA
#> 2:          NA            NA
#> 3:          NA            NA
#> 4:          NA            NA
#> 5:          NA            NA
#> 6:          NA            NA

# OpenMS - TMT data
feature_columns_tmt = c("PeptideSequence", "PrecursorCharge")
openms_processed = MSstatsPreprocess(
openms_cleaned,
NULL,
feature_columns_tmt,
remove_shared_peptides = TRUE,
remove_single_feature_proteins = TRUE,
feature_cleaning = list(remove_features_with_few_measurements = TRUE,
summarize_multiple_psms = max)
)
#>             ProteinName                               PeptideSequence
#> 1: sp|Q60854|SPB6_MOUSE .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR
#> 2: sp|Q60854|SPB6_MOUSE .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR
#> 3: sp|Q60854|SPB6_MOUSE .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR
#> 4: sp|Q60854|SPB6_MOUSE .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR
#> 5: sp|Q60854|SPB6_MOUSE .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR
#> 6: sp|Q60854|SPB6_MOUSE .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR
#>    PrecursorCharge                                             PSM Condition
#> 1:               3 .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR_3   Long_HF
#> 2:               3 .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR_3   Long_HF
#> 3:               3 .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR_3   Long_HF
#> 4:               3 .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR_3   Long_HF
#> 5:               3 .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR_3   Long_LF
#> 6:               3 .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR_3    Long_M
#>    BioReplicate   Run Channel Intensity Mixture TechRepMixture Fraction
#> 1:           21 3_3_3       1        NA       3            3_3        3
#> 2:           24 3_3_3       4        NA       3            3_3        3
#> 3:           26 3_3_3       6 1820.0721       3            3_3        3
#> 4:           28 3_3_3       8  445.7412       3            3_3        3
#> 5:           25 3_3_3       5 1580.9510       3            3_3        3
#> 6:           23 3_3_3       3 1508.3302       3            3_3        3

Annotation is created via the MSstatsMakeAnnotation function. It takes the cleaned dataset and annotation file as input. Additionally, key-value pairs can be passed to this function to change column names (not including dots and other symbols) in the annotation from names given by values to names given by keys.

For programmatic applications and consistency of the interface, filtering is done with the help of lists.

For filtering based on numerical scores (for example q-value filtering), the list should consist of elements named

• score_column: name of a column that stores the score,
• score_threshold: value above or below which measurements should be kept,
• direction: if “greater”, values greater than score_threshold will be kept; if “smaller”, values smaller than score_threshold will be kept;
• behavior: if “remove”, rows not below/above the threshold will be removed; if “replace”, intensity in rows not below/above the threshold will be replaced by a given value,
• handle_na: if “keep”, NA in the score column will not be removed,
• fill_value: value that will be used if behavior = "replace",
• filter: if TRUE, filtering will be performed (can be used for conditional filtering),
• drop_column: if TRUE, column that stored the score will be removed.

For example, to remove intensities smaller than 1, we could pass the following list to the score_filtering parameters:

list(
list(score_column = "Intensity", score_threshold = 1,
direction = "greater", behavior = "remove",
handle_na = "remove", fill_value = NA, filter = TRUE, drop = FALSE
)
)

For filtering based on patterns (for example, removing oxidation peptides), the list should consist of elements named

• col_name: name of a column that values that may be removed,
• filter_symbols: vector of values - rows with these values in col_name will be removed or corresponding intensities will be replaced,
• behavior: if “remove”, rows that contain filter_symbols in col_name will be removed; if “replace”, intensity in rows that contain filter_symbols in col_name will be replaced by a given value,
• fill_value: value that will be used if behavior = "replace",
• filter: if TRUE, filtering will be performed (can be used for conditional filtering),
• drop_column: if TRUE, column that stored the score will be removed.

For filtering based on exact values (for example, removing iRT proteins), the list should consists of elements named

• col_name: name of a column that stores strings that will be searched for given patterns,
• pattern: vector of regular expressions - rows with matching values in col_name will be removed,
• filter: if TRUE, filtering will be performed (can be used for conditional filtering),
• drop_column: if TRUE, column that stored the values for filtering will be removed.

# Fractions and balanced design

Finally, after preprocessing, MSstatsBalancedDesign function can be applied to handle fractions and create balanced design. For label-free and SRM data, it means that fractionation or technical replicates will be detected if these information is not provided. Features measured in multiple fractions (overlapped) will be assigned to a unique fraction. Then, the data will be adjusted so that within each fraction, every feature has a row for certain run. If the intensity value is missing, it will be denoted by NA.

For TMT data, a unique fraction will be selected for each overlapped feature and the data will adjusted so that within each run, every feature has a row for each channel. If the intensity is missing for a channel, it will be denoted by NA.

maxquant_balanced = MSstatsBalancedDesign(maxquant_processed, feature_columns)
#>   ProteinName PeptideSequence PrecursorCharge FragmentIon ProductCharge
#> 1      P06959     AEAPAAAPAAK               2          NA            NA
#> 2      P06959     AEAPAAAPAAK               2          NA            NA
#> 3      P06959     AEAPAAAPAAK               2          NA            NA
#> 4      P06959     AEAPAAAPAAK               2          NA            NA
#> 5      P06959     AEAPAAAPAAK               2          NA            NA
#> 6      P06959     AEAPAAAPAAK               2          NA            NA
#>   IsotopeLabelType Condition BioReplicate
#> 1                L         1            1
#> 2                L         1            1
#> 3                L         1            1
#> 4                L         2            2
#> 5                L         2            2
#> 6                L         2            2
#>                                           Run Fraction Intensity
#> 1 121219_S_CCES_01_01_LysC_Try_1to10_Mixt_1_1        1   4023100
#> 2 121219_S_CCES_01_02_LysC_Try_1to10_Mixt_1_2        1   5132500
#> 3 121219_S_CCES_01_03_LysC_Try_1to10_Mixt_1_3        1   2761600
#> 4 121219_S_CCES_01_04_LysC_Try_1to10_Mixt_2_1        1   2932900
#> 5 121219_S_CCES_01_05_LysC_Try_1to10_Mixt_2_2        1   4091800
#> 6 121219_S_CCES_01_06_LysC_Try_1to10_Mixt_2_3        1   4727000
dim(maxquant_balanced)
#> [1] 690  11
dim(maxquant_processed)
#> [1] 625  14

openms_balanced = MSstatsBalancedDesign(openms_processed, feature_columns_tmt)
#>            ProteinName                               PeptideSequence
#> 1 sp|Q60854|SPB6_MOUSE .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR
#> 2 sp|Q60854|SPB6_MOUSE .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR
#> 3 sp|Q60854|SPB6_MOUSE .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR
#> 4 sp|Q60854|SPB6_MOUSE .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR
#> 5 sp|Q60854|SPB6_MOUSE .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR
#> 6 sp|Q60854|SPB6_MOUSE .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR
#>   PrecursorCharge                                             PSM Mixture
#> 1               3 .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR_3       3
#> 2               3 .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR_3       3
#> 3               3 .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR_3       3
#> 4               3 .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR_3       3
#> 5               3 .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR_3       3
#> 6               3 .(TMT6plex)AFVEVNEEGTEAAAATAGMM(Oxidation)TVR_3       3
#>   TechRepMixture   Run Channel BioReplicate Condition Intensity
#> 1            3_3 3_3_3       1           21   Long_HF        NA
#> 2            3_3 3_3_3       2           22      Norm  1068.580
#> 3            3_3 3_3_3       3           23    Long_M  1508.330
#> 4            3_3 3_3_3       4           24   Long_HF        NA
#> 5            3_3 3_3_3       5           25   Long_LF  1580.951
#> 6            3_3 3_3_3       6           26   Long_HF  1820.072
dim(openms_balanced)
#> [1] 330  11
dim(openms_processed)
#> [1] 370  15

MSstatsBalancedDesign output is a data.frame of class MSstatsValidated. Such a data.frame will be recognized by statistical processing functions from MSstats and MSstatsTMT packages as a valid input, which will allow them to skip checks and transformation necessary to fit data into this format.