nPOP (Leduc et al. 2022) is an upgrade of the SCoPE2 protocole (Specht et al. 2021 and Petelski et al. 2021), where the mPOP sample preparation method is replaced by the nPOP method. nPOP processes samples using the Cellenion dispensing device and uses DMSO as lysis reagent instead of a freeze-thaw procedure. They also include the prioritized data acquisition mode as described by Huffman et al. 2022.

Let’s first load the replication package to make use of some helper functions. Those functions are only meant for this replication vignette and are not designed for general use.


scp and the SCoPE2 workflow

The code provided along with the article can be retrieved from this GitHub repository. The objective of this vignette is to replicate the analysis script while providing standardized, easy-to-read, and well documented code. Therefore, our first contribution is to formalize the data processing into a conceptual flow chart.

Overview of the processing workflow by Leduc et al.

Overview of the processing workflow by Leduc et al.

This replication vignette relies on a data framework dedicated to SCP data analysis that combines two Bioconductor classes (Vanderaa et al. 2021):

  • The SingleCellExperiment class provides an interface to many cutting edge methods for single-cell analysis
  • The QFeatures class facilitates manipulation and processing of MS-based quantitative data.

The scp vignette provides detailed information about the data structure. The scp package extends the functionality of QFeatures for single-cell application. scp offers a standardized implementation for single-cell processing methods.

The required packages for running this workflow are listed below.

## Core packages of this workflow
## Utility packages for data manipulation and visualization

scpdata and the leduc2022 dataset

We also implemented a data package called scpdata (@Vanderaa2022-qv). It distributes published SCP datasets, such as the leduc2022 dataset. The datasets were downloaded from the data source provided in the publication and formatted to a QFeatures object so that it is compatible with our software. The underlying data storage is based on the ExperimentHub package that provides a cloud-based storage infrastructure.

The leduc2022 dataset is provided at different levels of processing:

  • The raw data files that were generated by the mass-spectrometer software. This data is not included in scpdata.
  • A PSM data table obtained from the MaxQuant software that performs spectrum identification and quantification. PSM stands for peptide to spectrum match where MS spectra could successfully be assigned to peptide sequences. The provided PSM table was further processed using DART-ID (@Chen2019-uc) to improve the identification rate.
  • Peptide data are provided as intermediate output tables from the data processing. There are 2 tables: peptides normalized by reference and peptides further normalized (median centering of columns and rows) and log-transformed.
  • A protein data are provided as intermediate and final output tables from the data processing. There are 2 tables: proteins column and row centered to median and log2 transformed and proteins after imputation, batch correction and final column and row centering.

The workflow starts with the PSM table and will generate the peptide and the protein data. The authors provided the PSM dataset as a tabular text file called ev_updated.txt. Peptide and protein data are shared as CSV files. We highly value the effort the authors have made to publicly share all the data generated in their project, from raw files to final expression tables (see the Slavov Lab website).

We formatted the leduc2022 dataset following our data framework. The formatted data can be retrieved from the scpdata package using the leduc2022() function. All datasets in scpdata are called after the first author and the date of publication.

leduc <- leduc2022()

The data contain 138 different SingleCellExperiment objects that we refer to as assays. Each assay contains expression data along with feature metadata. Each row in an assay represents a feature that can either be a PSM, a peptide or a protein depending on the assay. Each column in an assay represents a sample. In the leduc object, samples are pooled using TMT-pro18 labeling, hence each assay contains 18 columns. Most samples are single-cells, but some samples are negative controls, references, carriers,… Below, we show the overview of the leduc object

## An instance of class QFeatures containing 138 assays:
##  [1] eAL00219: SingleCellExperiment with 6269 rows and 18 columns 
##  [2] eAL00220: SingleCellExperiment with 6603 rows and 18 columns 
##  [3] eAL00221: SingleCellExperiment with 6511 rows and 18 columns 
##  ...
##  [136] peptides_log: SingleCellExperiment with 12284 rows and 1543 columns 
##  [137] proteins_norm2: SingleCellExperiment with 2844 rows and 1543 columns 
##  [138] proteins_processed: SingleCellExperiment with 2844 rows and 1543 columns

134 out of the 138 assays are PSM data, each assay corresponding to a separate MS run. Notice that the assays were acquired in 2 sample preparation and chromatographic batches.

table(LcBatch = leduc$lcbatch,
      SamplePrepBatch = sub("AL.*", "", leduc$Set))
##        SamplePrepBatch
## LcBatch    e    w
##       A    0 1548
##       C  864    0

The dataset also contains a peptides, peptides_log, proteins_norm and proteins_processed assay. Those were provided by the authors. The objective of this vignette is to replicate these assays from the 134 PSM assays following the same procedure as the original script but using standardized functionality.

We extract these latter assays and keep them for later benchmarking. Using double brackets [[...]] extracts the desired assay as a SingleCellExperiment object. On the other hand, using simple brackets [row, col, assay] subsets the desired elements/assays but preserves the QFeatures data structure.

peptides_leduc <- leduc[["peptides"]]
peptides_log_leduc <- leduc[["peptides_log"]]
proteins_norm_leduc <- leduc[["proteins_norm2"]]
proteins_processed_leduc <- leduc[["proteins_processed"]]
leduc <- leduc[, , -(135:138)]
## Warning: 'experiments' dropped; see 'metadata'

We will compare the replications by comparing the set of filtered features (peptides or proteins) and samples. This is performed using this function.

compareSets <- function(setleduc, setscp) {
    allElements <- unique(c(setleduc, setscp))
    table(leduc2022 = allElements %in% setleduc,
          scp = allElements %in% setscp)

We will also compare the replication based on the quantitative data. We again create a dedicated function to perform this.

compareQuantitativeData <- function(sceleduc, scescp) {
    rows <- intersect(rownames(sceleduc),
    cols <- intersect(colnames(sceleduc),
    err <- assay(sceleduc)[rows, cols] - assay(scescp)[rows, cols]
    data.frame(difference = as.vector(err[!])) %>%
        ggplot() +
        aes(x = difference) +
        geom_histogram(bins = 50) +
        xlab("nPOP - scp") +
        scale_y_continuous(labels = scales::scientific) +

PSM filtering

After importing the data, Leduc et al. filter low-confidence PSMs. Each PSM assay contains feature meta-information that are stored in the assay rowData. The QFeatures package allows to quickly filter the rows of an assay by using these information. The available variables in the rowData are listed below for each assay.

## CharacterList of length 134
## [["eAL00219"]] Sequence Length ... Leading.razor.protein.symbol
## [["eAL00220"]] Sequence Length ... Leading.razor.protein.symbol
## [["eAL00221"]] Sequence Length ... Leading.razor.protein.symbol
## [["eAL00222"]] Sequence Length ... Leading.razor.protein.symbol
## [["eAL00223"]] Sequence Length ... Leading.razor.protein.symbol
## [["eAL00224"]] Sequence Length ... Leading.razor.protein.symbol
## [["eAL00225"]] Sequence Length ... Leading.razor.protein.symbol
## [["eAL00226"]] Sequence Length ... Leading.razor.protein.symbol
## [["eAL00227"]] Sequence Length ... Leading.razor.protein.symbol
## [["eAL00228"]] Sequence Length ... Leading.razor.protein.symbol
## ...
## <124 more elements>

Remove contaminant, noisy and low-confidence spectra

We first remove spectra that are matched to contaminant proteins and reverse hits. We also remove PSMs that have been matched from impure spectra, that are spectra containing co-eluting peptides. These are identified based on the parental ion fraction (PIF), computed by MaxQuant. Finally, we also want to remove PSM with poor matching confidence, as defined by the false discovery rate (FDR) computed by DART-ID.

We can extract the information from the rowData of several assays using the rbindRowData function. It takes the rowData of interest and returns a single DataFrame table with variables of interest. We extract such a table for the different variables listed above to create a quality control plot.

rd <- data.frame(rbindRowData(leduc, i = names(leduc)))
ggplot(rd) +
    aes(x = dart_PEP) +
    geom_histogram() +
    geom_vline(xintercept = 0.01) +
    ggplot(rd) +
    aes(x = PIF) +
    geom_histogram() +
    geom_vline(xintercept = 0.6)
## Warning: Removed 181437 rows containing non-finite values (stat_bin).

We next remove the PSMs that are matched to potential contaminants (Potential.contaminant is + and Proteins starts with CON), reverse hits (Reverse is + and Leading.razor.protein starts with REV), noisy spectra (PIF is missing or greater than 0.6) and low-confidence spectra with at 1% FDR threshold (dart_qval smaller than 0.01). We can perform this on our QFeatures object using the filterFeatures() function. The different pieces of information are directly accessed from the rowData of each assay.

leduc <- filterFeatures(leduc, ~ Potential.contaminant != "+" &
                            !grepl("CON", Proteins) &
                            Reverse != "+" &
                            !grepl("REV", Leading.razor.protein) &
                            ( | PIF > 0.6) &
                            dart_qval < 0.01)

Sample to carrier filter

The PSMs are next filtered based on the sample to carrier ratio (SCR), that is the TMT ion intensity of a single-cell sample divided by the TMT ion intensity of the carrier (200 cell equivalent) acquired during the same run as the sample. It is expected that the carrier intensities are much higher than the single-cell intensities. We implemented the computeSCR() function that computes the SCR for each PSM averaged over all samples of interest in a given assay. A PSM is removed when the mean SCR exceeds 10 %. To perform this, we need to tell the function which columns are the samples of interest and which column is the carrier. The colData of the QFeatures object is used to define this.

##       Carrier Melanoma cell      Monocyte    NegControl     Reference 
##           134           878           877           120           134 
##        Unused 
##           269

In this dataset, SampleType gives the type of sample that is present in each TMT channel. There 5 types of samples:

  • The carrier channels (Carrier) contain 200 cell equivalents and are meant to boost the peptide identification rate.
  • The normalization channels (Reference) are used to partially correct for between-run variation.
  • The unused channels (Unused) are channels that are left empty due to isotopic cross-contamination.
  • The negative controls (NegControl) contain samples that do not contain any cell but are processed as single-cell samples.
  • The single-cell sample channels contain the single-cell samples of interest (Melanoma cell or Monocyte).

The computeSCR function expects the user to provide a pattern (following regular expression syntax) that uniquely identifies a carrier channel in each run and the samples or blanks. The function will store the mean SCR of each feature in the rowData of each assay.

leduc <- computeSCR(leduc, names(leduc),
                    colvar = "SampleType", 
                    samplePattern = "Mel|Macro",
                    carrierPattern = "Carrier",
                    sampleFUN = "mean",
                    rowDataName = "MeanSCR")

Before applying the filter, we plot the distribution of the mean SCR.

rbindRowData(leduc, i = names(leduc)) %>%
    data.frame %>%
    ggplot(aes(x = MeanSCR)) +
    geom_histogram() +
    geom_vline(xintercept = 0.1) +

A great majority of the PSMs have a mean SCR that is lower than 10%, as expected. Since the mean SCR is stored in the rowData, we can apply filterFeatures() on the object to remove PSMs with high average SCR.

leduc <- filterFeatures(leduc, ~ 
                            ! & !is.infinite(MeanSCR) &
                            MeanSCR < 0.05)

Filter on summed single-cell signal

Finally, we remove PSM that have no signal in single-cell samples. This is not explicitely implemented in scp. To add custom information to rowData, you need to provide a list of DataFrames. The name of the elements in the list should correspond to the names of the assays where the rowData is modified. The column names of the DataFrame indicate which variable should be modified or added (if they do not exist yet). So, for each assay, we compute the summed signal in single-cells (and negative controls) and store the results in a DataFrame.

sums <- lapply(names(leduc), function(i) {
    sce <- leduc[[i]]
    sel <- grep("Mel|Macro|Neg", colData(leduc)[colnames(sce), "SampleType"])
    x <- assay(sce)[, sel, drop = FALSE]
    rs <- rowSums(x, na.rm = TRUE)
    DataFrame(ScSums = rs)

The list of DataFrame is named after the corresponding assays and the rowData of the leduc object is modified.

names(sums) <- names(leduc)
rowData(leduc) <- sums

To verify this new piece of data was correctly added, we plot the summed signal for each PSM.

rbindRowData(leduc, i = names(leduc)) %>%
    data.frame %>%
    ggplot(aes(x = ScSums)) +
    geom_histogram() +

We apply the final filter using filterFeatures().

leduc <- filterFeatures(leduc, ~ ScSums != 0)

Normalize to reference

In order to partially correct for between-run variation, Leduc et al. compute relative reporter ion intensities. This means that intensities measured for single-cells are divided by the reference channel. We use the divideByReference() function that divides channels of interest by the reference channel. Similarly to computeSCR, we can point to the samples and the reference columns in each assay using the annotation contained in the colData. We will here divide all columns (using the regular expression wildcard .) by the reference channel (Reference).

leduc <- divideByReference(leduc, i = names(leduc),
                           colvar = "SampleType", 
                           samplePattern = ".",
                           refPattern = "Reference")

Notice that when taking all samples we also include the reference channel itself. Hence, from now on, the reference channels will contain only ones.

Aggregate PSM data to peptide data

Now that the PSM assays are processed, we can aggregate them to peptides. This is performed using the aggregateFeaturesOverAssays() function. This is a wrapper function in scp that sequentially calls the aggregateFeatures from the QFeatures package over the different assays. For each assay, the function aggregates several PSMs into a unique peptide given an aggregating variable in the rowData (peptide sequence) and a user-supplied aggregating function (the median for instance). Regarding the aggregating function, the original analysis removes duplicated peptide sequences per run by taking the first non-missing value. While better alternatives are documented in QFeatures::aggregateFeatures, we still use this approach for the sake of replication and for illustrating that custom functions can be applied.

remove.duplicates <- function(x)
    apply(x, 2, function(xx) xx[which(![1]] )

The aggregated peptide assays must be given a name. We here used the original names with peptides_ at the start.

peptideAssays <- paste0("peptides_", names(leduc))

We now have all the required information to aggregate the PSMs in the different batches to peptides.

leduc <- aggregateFeaturesOverAssays(leduc,
                                     i = names(leduc),
                                     fcol = "modseq",
                                     name = peptideAssays,
                                     fun = remove.duplicates)

Under the hood, the QFeatures architecture preserves the relationship between the aggregated assays. See ?AssayLinks for more information on relationships between assays. Notice that aggregateFeaturesOverAssays created as many new assays as the number of supplied assays.

## An instance of class QFeatures containing 268 assays:
##  [1] eAL00219: SingleCellExperiment with 3555 rows and 18 columns 
##  [2] eAL00220: SingleCellExperiment with 3981 rows and 18 columns 
##  [3] eAL00221: SingleCellExperiment with 3785 rows and 18 columns 
##  ...
##  [266] peptides_wAL00284: SingleCellExperiment with 3211 rows and 18 columns 
##  [267] peptides_wAL00285: SingleCellExperiment with 3277 rows and 18 columns 
##  [268] peptides_wAL00286: SingleCellExperiment with 3411 rows and 18 columns

Join assays

Up to now, we kept the data belonging to each MS run in separate assays. We now combine all batches into a single assay. This can easily be done using the joinAssays() function from the QFeatures package.

Consensus mapping of peptides to proteins

We need to account for an issue in the data. joinAssays() will only keep the metadata variables that have the same value between matching rows. However, some peptide sequences map to one protein in one run and to another protein in another run. Hence, the protein sequence is not constant for all peptides and is removed during joining. It is important we keep the protein sequence in the rowData since we will later need it to aggregate peptides to proteins. To avoid this issue, we replace the problematic peptides to protein mappings through a majority vote.

## Generate a list of DataFrames with the information to modify
rbindRowData(leduc, i = grep("^pep", names(leduc))) %>%
    data.frame %>%
    group_by(modseq) %>%
    ## The majority vote happens here
    mutate(Leading.razor.protein.symbol =
                          decreasing = TRUE))[1]) %>%
    select(modseq, Leading.razor.protein.symbol) %>%
    filter(!duplicated(modseq, Leading.razor.protein.symbol)) ->
consensus <- lapply(peptideAssays, function(i) {
    ind <- match(rowData(leduc[[i]])$modseq, ppMap$modseq)
    DataFrame(Leading.razor.protein.symbol =
## Name the list
names(consensus) <- peptideAssays
## Modify the rowData
rowData(leduc) <- consensus

Cleaning missing data

Another important step before we join the assays is to replace zero and infinite values by NAs. The zeros can be biological zeros or technical zeros and differentiating between the two types is a difficult task, they are therefore better considered as missing. The infinite values arose during the normalization by the reference because the channel values are divide by a zero from the reference channel. This artefact could easily be avoided if we had replace the zeros by NAs at the beginning of the workflow, what we strongly recommend for future analyses.

The infIsNA() and the zeroIsNA() functions automatically detect infinite and zero values, respectively, and replace them with NAs. Those two functions are provided by the QFeatures package.

leduc <- infIsNA(leduc, i = peptideAssays)
leduc <- zeroIsNA(leduc, i = peptideAssays)

Join assays

Now that the peptides are correctly matched to proteins and missing values are correctly formatted, we can join the assays.

leduc <- joinAssays(leduc,
                    i = peptideAssays,
                    name = "peptides")

joinAssays has created a new assay called peptides that combines the previously aggregated peptide assays.

## An instance of class QFeatures containing 269 assays:
##  [1] eAL00219: SingleCellExperiment with 3555 rows and 18 columns 
##  [2] eAL00220: SingleCellExperiment with 3981 rows and 18 columns 
##  [3] eAL00221: SingleCellExperiment with 3785 rows and 18 columns 
##  ...
##  [267] peptides_wAL00285: SingleCellExperiment with 3277 rows and 18 columns 
##  [268] peptides_wAL00286: SingleCellExperiment with 3411 rows and 18 columns 
##  [269] peptides: SingleCellExperiment with 20480 rows and 2412 columns

Filter single-cells based on median CV

Leduc et al. proceed with filtering the single-cells. The filtering is mainly based on the median coefficient of variation (CV) per cell. The median CV measures the consistency of quantification for a group of peptides that belong to a protein. We remove cells that exhibit high median CV over the different proteins. We compute the median CV per cell using the medianCVperCell() function from the scp package. The function takes the protein information from the rowData of the assays that will tell how to group the features (peptides) when computing the CV. Note that we supply the peptide assays before joining in a single assays (i = peptideAssays). This is because SCoPE2 performs a custom normalization (norm = "SCoPE2"). Each row in an assay is normalized by a scaling factor. This scaling factor is the row mean after dividing the columns by the median. The authors retained CVs that are computed using at least 3 peptides (nobs = 3).

leduc <- medianCVperCell(leduc,
                         i = peptideAssays,
                         groupBy = "Leading.razor.protein.symbol",
                         nobs = 3,
                         na.rm = TRUE,
                         colDataName = "MedianCV",
                         norm = "SCoPE2")
## Warning in medianCVperCell(leduc, i = peptideAssays, groupBy = "Leading.razor.protein.symbol", : The median CV could not be computed for one or more samples. You may want to try a smaller value for 'nobs'.

The computed CVs are stored in the colData. We can now filter cells that have reliable quantifications. The negative controls are not expected to have reliable quantifications and hence can be used to estimate a null distribution of the CV. This distribution helps defining a threshold that filters out single-cells that contain noisy quantification.

colData(leduc) %>% 
    data.frame %>% 
    filter(grepl("Mono|Mel|Neg", SampleType)) %>% 
    mutate(control = ifelse(grepl("Neg", SampleType), "ctl", "sc")) %>% 
    ggplot() +
    aes(x = MedianCV,
        fill = control) + 
    geom_density(alpha = 0.5, adjust = 1) +
    geom_vline(xintercept = 0.42) +
    xlim(0.2, 0.8) +
    theme_minimal() +
    scale_fill_manual(values = c( "black", "purple2")) + 
    xlab("Quantification variability") + 
    ylab("Fraction of cells")
## Warning: Removed 31 rows containing non-finite values (stat_density).

We can see that the protein quantification for single-cells are much more consistent within single-cell channels than within blank channels. A threshold of 0.42 best separates single-cells from empty channels.

We keep the cells that pass the median CV threshold. Furthermore, we keep melanoma cells and monocytes as those represent the samples of interest. We can extract the sample names that pass the CV and sample type filters using the subsetByColData() function.

leduc <- 
                    !$MedianCV) &
                        leduc$MedianCV < 0.42 &
                        grepl("Mono|Mel", leduc$SampleType))

Compare intermediate results

At this stage of the processing, the last assay should be similar to the peptides_leduc data provided by the authors. Let’s compare the filtered cells.

##          scp
## leduc2022 FALSE TRUE
##     FALSE     0   27
##     TRUE      7 1549

There is an excellent agreement between the the original and the replicated vignette. Let’s do the same for the filtered peptides.

##          scp
## leduc2022 FALSE  TRUE
##     FALSE     0    27
##     TRUE    351 20453

Finally let’s compare the quantitative data

compareQuantitativeData(peptides_leduc, leduc[["peptides"]])

The replication is close to perfect. Note however that this vignette is more stringent with respect to the number of selected peptides. We cannot explain this difference.


The columns (samples) then the rows (peptides) are normalized by dividing the relative intensities by the median relative intensities. The column normalization is implemented as the normalize() function with the argument method = div.median. The row normalization is not available from normalizet(), but is easily performed using the sweep function from the QFeatures package that is inspired from the base::sweep function.

## Scale column with median
leduc <- normalize(leduc,
                   i = "peptides",
                   method = "div.median",
                   name = "peptides_norm1")
## Scale rows with median
leduc <- sweep(leduc,
               i = "peptides_norm1",
               name = "peptides_norm2",
               MARGIN = 1,
               FUN = "/",
               STATS = rowMedians(assay(leduc[["peptides_norm1"]]),
                                  na.rm = TRUE))

Each normalization step is stored in a separate assay. An important aspect to note here is that

Missing data filtering

Peptides that contain many missing values are not informative. Therefore, the authors remove those with more than 99 % missing data. This is done using the filterNA() function from QFeatures.

leduc <- filterNA(leduc,
                  i = "peptides_norm2",
                  pNA = 0.99)

They also remove cells with more than 99 % missing data. This is performed by first computing the amount of missing data in the assay using nNA(). We then subset the cells that meet the criterion.

nnaRes <- nNA(leduc, "peptides_norm2")
sel <- nnaRes$nNAcols$pNA < 99
leduc[["peptides_norm2"]] <- leduc[["peptides_norm2"]][, sel]
## Warning in replaceAssay(x = x, y = value, i = i): Links between assays were
## lost/removed during replacement. See '?addAssayLink' to restore them manually.


Peptide data is log2-transformed before aggregating to proteins. This is performed by the logTransform() function from QFeatures.

leduc <- logTransform(leduc,
                      base = 2,
                      i = "peptides_norm2",
                      name = "peptides_log")

Compare intermediate results

At this stage of the processing, the last assay should be similar to the peptides_log_leduc data provided by the authors. Let’s compare the filtered cells.

##          scp
## leduc2022 FALSE TRUE
##     FALSE     0   27
##     TRUE      2 1541

There is an excellent agreement between the the original and the replicated vignette. Let’s do the same for the filtered peptides.

##          scp
## leduc2022 FALSE  TRUE
##     FALSE     0    18
##     TRUE     51 12233

Notice here that most peptides that this vignette removed earlier are now also removed by the original analysis. There is an excellent agreement as well between selected peptides. Finally let’s compare the quantitative data.

compareQuantitativeData(peptides_log_leduc, leduc[["peptides_log"]])

The agreement is still very good, with a sharp peak around 0. However, we can see that the range of differences starts to increase, probably because numerical differences propagate as we progress through the data processing.

Aggregate peptide data to protein data

Similarly to aggregating PSM data to peptide data, we can aggregate peptide data to protein data using the aggregateFeatures function. Note that we here use the median as a summarizing function.

leduc <- aggregateFeatures(leduc,
                           i = "peptides_log",
                           name = "proteins",
                           fcol = "Leading.razor.protein.symbol",
                           fun = matrixStats::colMedians, 
                           na.rm = TRUE)


Normalization is performed similarly to peptide normalization. We use the same functions, but since the data were log-transformed at the peptide level, we subtract by the median instead of dividing.

## Center columns with median
leduc <- normalize(leduc,
                   i = "proteins",
                   method = "center.median",
                   name = "proteins_norm1")
## Scale rows with median
leduc <- sweep(leduc,
               i = "proteins_norm1",
               name = "proteins_norm2",
               MARGIN = 1,
               FUN = "-",
               STATS = rowMedians(assay(leduc[["proteins_norm1"]]),
                                  na.rm = TRUE))

Compare intermediate results

At this stage of the processing, the last assay should be similar to the proteins_norm_leduc data provided by the authors. Let’s compare the filtered cells.

##          scp
## leduc2022 FALSE TRUE
##     FALSE     0   27
##     TRUE      2 1541

There is an excellent agreement between the the original and the replicated vignette. Let’s do the same for the filtered proteins.

##          scp
## leduc2022 FALSE TRUE
##     FALSE     0 2837
##     TRUE   2844    0

There is an almost perfect agreement between the selected proteins. selected Finally let’s compare the quantitative data.

compareQuantitativeData(proteins_norm_leduc, leduc[["proteins_norm2"]])

Again, there is a very sharp peak around 0.


The protein data is majorily composed of missing values. The graph below shows the distribution of the proportion missingness in cells. Cells contain on average 65 % missing values.

data.frame(pNA = nNA(leduc, "proteins_norm2")$nNAcols$pNA) %>%
    ggplot(aes(x = pNA)) +
    geom_histogram() +
    xlab("Percentage missingnes per cell")

The missing data is imputed using K nearest neighbors. The authors run KNN with k = 3. We made a wrapper around the author’s code to apply imputation to our QFeatures object.

leduc <- imputeKnnSCoPE2(leduc,
                         i = "proteins_norm2",
                         name = "proteins_impd",
                         k = 3)

QFeatures provides the impute function that serves as an interface to different imputation algorithms among which the KNN algorithm from impute::impute.knn. However, the KNN implementation in the oringal analysis and in impute.knn are different. Leduc et al. perform KNN imputation in the sample space, meaning that data from neighbouring cells are used to impute the central cell, whereas impute::impute.knn performs KNN imputation in the feature space, meaning that data from neighbouring features are used to impute the missing values from the central features. We provide the code for KNN imputation with QFeatures but do not run in order to replicate the original analysis.

leduc <- impute(leduc,
                i = "proteins_norm2",
                method = "knn",
                k = 3, rowmax = 1, colmax= 1,
                maxp = Inf, rng.seed = 1234)

Batch correction

The next step is to correct for the remaining batch effects. The data were acquired as a series of MS runs. Recall we had 134 assays at the beginning of the workflow. Each MS run can be subjected to technical perturbations that lead to differences in the data. Furthermore, TMT labeling can also influence the quantification. These effects must be accounted for to avoid attributing biological effects to technical effects. The limma algorithm (CITE-Ritchie) is used by Leduc et al. to correct for batch effects. It can take up to 2 batch variables, in this case the MS acquisition batch and the TMT channel, while protecting for variables of interest, the sample type in this case. All the information is contained in the colData of the QFeatures object. We first extract the assays with the associated colData.

sce <- getWithColData(leduc, "proteins_impd")
## Warning: 'experiments' dropped; see 'metadata'

We next create the design matrix. We then perform the batch correction and overwrite the data matrix. Recall the data matrix can be accessed using the assay function.

model <- model.matrix(~ SampleType, data = colData(sce))
assay(sce) <- removeBatchEffect(x = assay(sce),
                                batch = sce$lcbatch,
                                batch2 = sce$Channel,
                                design = model)

Finally, we add the batch corrected assay to the QFeatures object and create the feature links.

leduc <- addAssay(leduc, y = sce, name = "proteins_batchC")
leduc <- addAssayLinkOneToOne(leduc, from = "proteins_impd",
                              to = "proteins_batchC")


The very last step of the data processing workflow is a new round of normalization.

## Center columns with median
leduc <- normalize(leduc,
                   i = "proteins_batchC",
                   method = "center.median",
                   name = "proteins_batchC_norm1")
## Scale rows with median
leduc <- sweep(leduc,
               i = "proteins_batchC_norm1",
               name = "proteins_processed",
               MARGIN = 1,
               FUN = "-",
               STATS = rowMedians(assay(leduc[["proteins_batchC_norm1"]]),
                                  na.rm = TRUE))

Compare the final results

At the end of the processing, the last assay should be similar to the proteins_processed_leduc data provided by the authors. Let’s compare the filtered cells.

##          scp
## leduc2022 FALSE TRUE
##     FALSE     0   27
##     TRUE      2 1541

There is an excellent agreement between the selected cells from the original and this vignette. Let’s do the same for the filtered proteins.

##          scp
## leduc2022 FALSE TRUE
##     FALSE     0 2837
##     TRUE   2844    0

The agreement is also excellent between filtered proteins. Finally let’s compare the quantitative data


The differences are still sharply peaked around 0. However the differences are more spread and the range is larger compared to the previous steps.

Overall, we can see good replication of the data processing, although early differences seem to get amplified as we progress through the different processing steps. We next compare the dimension reduction results to get a more qualitative assessment.


We run the same PCA procedure as performed by the authors, that is a weighted PCA where the weight for a protein is defined as the summed correlation with the other proteins.

sce <- getWithColData(leduc, "proteins_processed")
## Warning: 'experiments' dropped; see 'metadata'
## Warning: Ignoring redundant column names in 'colData(x)':
pcaRes <- pcaSCoPE2(sce)
## Compute percent explained variance
pcaPercentVar <- round(pcaRes$values[1:2] / sum(pcaRes$values) * 100)
## Plot PCA
data.frame(PC = pcaRes$vectors[, 1:2],
           colData(sce)) %>%
    ggplot() +
    aes(x = PC.1, 
        y = PC.2, 
        colour = SampleType) +
    geom_point(alpha = 0.5) +
    xlab(paste0("PC1 (", pcaPercentVar[1], "%)")) +
    ylab(paste0("PC2 (", pcaPercentVar[2], "%)"))+
    ggtitle("PCA on scp processed protein data")

The PCA plot is very similar to the published PCA plot.

pcaResLeduc <- pcaSCoPE2(proteins_processed_leduc)
## Compute percent explained variance
pcaPercentVar <- round(pcaResLeduc$values[1:2] / sum(pcaResLeduc$values) * 100)
## Plot PCA
data.frame(PC = pcaResLeduc$vectors[, 1:2],
           colData(proteins_processed_leduc)) %>%
    ggplot() +
    aes(x = PC.1, 
        y = PC.2, 
        colour = SampleType) +
    geom_point(alpha = 0.5) +
    xlab(paste0("PC1 (", pcaPercentVar[1], "%)")) +
    ylab(paste0("PC2 (", pcaPercentVar[2], "%)")) +
    ggtitle("PCA on processed protein data by Leduc et al.")

Here again we can see the replicated PCA from this vignette is very similar to the PCA published by the authors.

Using standard PCA, we obtain the same cell patterns although the explained variance differs.

## Perform PCA, see ?runPCA for more info about arguments
runPCA(sce, ncomponents = 50,
       ntop = Inf,
       scale = TRUE,
       exprs_values = 1,
       name = "PCA") %>%
    ## Plotting is performed in a single line of code
    plotPCA(colour_by = "SampleType")


In this vignette, we have demonstrated that the scp package is able to accurately reproduce the analysis published by Leduc et al. We not only support the reliability of the published work, but we also offer a formalization and standardization of the pipeline by means of easy-to-read and highly documented code. This workflow can serve as a starting ground to improve upon the current methods and to design new modelling tools dedicated to single-cell proteomics.

Reproduce this vignette

You can reproduce this vignette using Docker:

docker pull cvanderaa/scp_replication_docker:v1
docker run \
    -e PASSWORD=bioc \
    -p 8787:8787 \

Open your browser and go to http://localhost:8787. The USER is rstudio and the password is bioc. You can find the vignette in the vignettes folder.

See the website home page for more information.


Hardware and software

The system details of the machine that built the vignette are:

## Machine: Linux (5.15.0-48-generic)
## R version: R.4.2.1 (svn: 82513)
## RAM: 16.5 GB
## CPU: 16 core(s) - 11th Gen Intel(R) Core(TM) i7-11800H @ 2.30GHz


The total time required to compile this vignette is:

## 21.28293 mins


The final leduc object size is:

## [1] "2.6 Gb"

Session info

## R version 4.2.1 (2022-06-23)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 20.04.4 LTS
## Matrix products: default
## BLAS:   /usr/lib/x86_64-linux-gnu/blas/
## LAPACK: /usr/lib/x86_64-linux-gnu/lapack/
## locale:
##  [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
##  [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8    
##  [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
##  [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
##  [9] LC_ADDRESS=C               LC_TELEPHONE=C            
## attached base packages:
## [1] stats4    stats     graphics  grDevices utils     datasets  methods  
## [8] base     
## other attached packages:
##  [1] benchmarkme_1.0.8           scater_1.25.7              
##  [3] scuttle_1.7.4               patchwork_1.1.2            
##  [5] forcats_0.5.2               stringr_1.4.1              
##  [7] dplyr_1.0.10                purrr_0.3.4                
##  [9] readr_2.1.3                 tidyr_1.2.1                
## [11] tibble_3.1.8                ggplot2_3.3.6              
## [13] tidyverse_1.3.2             limma_3.53.10              
## [15] SCP.replication_0.2.1       SingleCellExperiment_1.19.1
## [17] scpdata_1.5.4               ExperimentHub_2.5.0        
## [19] AnnotationHub_3.5.2         BiocFileCache_2.5.0        
## [21] dbplyr_2.2.1                scp_1.7.4                  
## [23] QFeatures_1.7.3             MultiAssayExperiment_1.23.9
## [25] SummarizedExperiment_1.27.3 Biobase_2.57.1             
## [27] GenomicRanges_1.49.1        GenomeInfoDb_1.33.7        
## [29] IRanges_2.31.2              S4Vectors_0.35.4           
## [31] BiocGenerics_0.43.4         MatrixGenerics_1.9.1       
## [33] matrixStats_0.62.0          BiocStyle_2.25.0           
## loaded via a namespace (and not attached):
##   [1] utf8_1.2.2                    reticulate_1.26              
##   [3] tidyselect_1.1.2              RSQLite_2.2.17               
##   [5] AnnotationDbi_1.59.1          grid_4.2.1                   
##   [7] BiocParallel_1.31.12          munsell_0.5.0                
##   [9] ScaledMatrix_1.5.1            codetools_0.2-18             
##  [11] ragg_1.2.3                    withr_2.5.0                  
##  [13] colorspace_2.0-3              filelock_1.0.2               
##  [15] highr_0.9                     knitr_1.40                   
##  [17] labeling_0.4.2                GenomeInfoDbData_1.2.9       
##  [19] bit64_4.0.5                   farver_2.1.1                 
##  [21] rprojroot_2.0.3               vctrs_0.4.2                  
##  [23] generics_0.1.3                xfun_0.33                    
##  [25] doParallel_1.0.17             R6_2.5.1                     
##  [27] ggbeeswarm_0.6.0              clue_0.3-61                  
##  [29] rsvd_1.0.5                    locfit_1.5-9.6               
##  [31] MsCoreUtils_1.9.1             AnnotationFilter_1.21.0      
##  [33] bitops_1.0-7                  cachem_1.0.6                 
##  [35] DelayedArray_0.23.2           assertthat_0.2.1             
##  [37] promises_1.2.0.1              scales_1.2.1                 
##  [39] googlesheets4_1.0.1           beeswarm_0.4.0               
##  [41] gtable_0.3.1                  beachmat_2.13.4              
##  [43] OrgMassSpecR_0.5-3            benchmarkmeData_1.0.4        
##  [45] sva_3.45.0                    rlang_1.0.6                  
##  [47] genefilter_1.79.0             systemfonts_1.0.4            
##  [49] splines_4.2.1                 lazyeval_0.2.2               
##  [51] gargle_1.2.1                  broom_1.0.1                  
##  [53] BiocManager_1.30.18           yaml_2.3.5                   
##  [55] modelr_0.1.9                  backports_1.4.1              
##  [57] httpuv_1.6.6                  tools_4.2.1                  
##  [59] bookdown_0.29                 ellipsis_0.3.2               
##  [61] jquerylib_0.1.4               Rcpp_1.0.9                   
##  [63] sparseMatrixStats_1.9.0       zlibbioc_1.43.0              
##  [65] RCurl_1.98-1.9                viridis_0.6.2                
##  [67] cowplot_1.1.1                 haven_2.5.1                  
##  [69] ggrepel_0.9.1                 cluster_2.1.4                
##  [71] fs_1.5.2                      magrittr_2.0.3               
##  [73] reprex_2.0.2                  googledrive_2.0.0            
##  [75] ProtGenerics_1.29.0           hms_1.1.2                    
##  [77] mime_0.12                     evaluate_0.16                
##  [79] xtable_1.8-4                  XML_3.99-0.11                
##  [81] readxl_1.4.1                  gridExtra_2.3                
##  [83] compiler_4.2.1                crayon_1.5.2                 
##  [85] htmltools_0.5.3               mgcv_1.8-40                  
##  [87] later_1.3.0                   tzdb_0.3.0                   
##  [89] lubridate_1.8.0               DBI_1.1.3                    
##  [91] MASS_7.3-58.1                 rappdirs_0.3.3               
##  [93] Matrix_1.5-1                  cli_3.4.1                    
##  [95] parallel_4.2.1                igraph_1.3.5                 
##  [97] pkgconfig_2.0.3               pkgdown_2.0.6                
##  [99] foreach_1.5.2                 xml2_1.3.3                   
## [101] annotate_1.75.0               vipor_0.4.5                  
## [103] bslib_0.4.0                   XVector_0.37.1               
## [105] rvest_1.0.3                   digest_0.6.29                
## [107] Biostrings_2.65.6             rmarkdown_2.16               
## [109] cellranger_1.1.0              edgeR_3.39.6                 
## [111] DelayedMatrixStats_1.19.1     curl_4.3.2                   
## [113] shiny_1.7.2                   lifecycle_1.0.2              
## [115] nlme_3.1-159                  jsonlite_1.8.2               
## [117] BiocNeighbors_1.15.1          desc_1.4.2                   
## [119] viridisLite_0.4.1             fansi_1.0.3                  
## [121] pillar_1.8.1                  lattice_0.20-45              
## [123] KEGGREST_1.37.3               fastmap_1.1.0                
## [125] httr_1.4.4                    survival_3.4-0               
## [127] interactiveDisplayBase_1.35.0 glue_1.6.2                   
## [129] iterators_1.0.14              png_0.1-7                    
## [131] BiocVersion_3.16.0            bit_4.0.4                    
## [133] stringi_1.7.8                 sass_0.4.2                   
## [135] BiocBaseUtils_0.99.12         blob_1.2.3                   
## [137] textshaping_0.3.6             BiocSingular_1.13.1          
## [139] memoise_2.0.1                 irlba_2.3.5.1


This vignette is distributed under a CC BY-SA licence licence.