# Grouping FTICR-MS data with xcms

Joachim Bargsten and Johannes Rainer

#### 19 June 2025

#### Package

xcms 4.7.2

# 1 Introduction

This document describes how to use xcms for the analysis of direct injection mass spec data, including peak detection, calibration and correspondence (grouping of peaks across samples).

# 2 Peak detection

Prior to any other analysis step, peaks have to be identified in the mass spec data. In contrast to the typical metabolomics workflow, in which peaks are identified in the chromatographic (time) dimension, in direct injection mass spec data sets peaks are identified in the m/z dimension. xcms uses functionality from the MassSpecWavelet package to identify such peaks.

Below we load the required packages. For information on the parallel processing setup please see the BiocParallel vignette.

    library(MSnbase)
    library(xcms)
    library(MassSpecWavelet)

    register(SerialParam())

In this documentation we use an example data set from the msdata package. Assuming that msdata is installed, we locate the path of the package and load the data set. We create also a `data.frame` describing the experimental setup based on the file names.

    mzML_path <- system.file("fticr-mzML", package = "msdata")
    mzML_files <- list.files(mzML_path, recursive = TRUE, full.names = TRUE)

    ## We're subsetting to 2 samples per condition
    mzML_files <- mzML_files[c(1, 2, 6, 7)]

    ## Create a data.frame assigning samples to sample groups, i.e. ham4 and ham5.
    grp <- rep("ham4", length(mzML_files))
    grp[grep(basename(mzML_files), pattern = "^HAM005")] <- "ham5"
    pd <- data.frame(filename = basename(mzML_files), sample_group = grp)

    ## Load the data.
    ham_raw <- readMSData(files = mzML_files,
                          pdata = AnnotatedDataFrame(pd),
                          mode = "onDisk")

The data files are from direct injection mass spectrometry experiments, i.e. we have only a single spectrum available for each sample and no retention times.

    ## Only a single spectrum with an *artificial* retention time is available
    ## for each sample
    rtime(ham_raw)

    ## F1.S1 F2.S1 F3.S1 F4.S1
    ##    -1    -1    -1    -1

Peaks are identified within each spectrum using the mass spec wavelet method.

    ## Define the parameters for the peak detection
    msw <- MSWParam(scales = c(1, 4, 9), nearbyPeak = TRUE, winSize.noise = 500,
                    SNR.method = "data.mean", snthresh = 10)

    ham_prep <- findChromPeaks(ham_raw, param = msw)

    head(chromPeaks(ham_prep))

    ##            mz    mzmin    mzmax rt rtmin rtmax    into     maxo       sn intf
    ## CP01 403.2367 403.2279 403.2447 -1    -1    -1 4735258 372259.4 22.97534   NA
    ## CP02 409.1845 409.1747 409.1936 -1    -1    -1 4158405 310572.1 20.61382   NA
    ## CP03 413.2677 413.2585 413.2769 -1    -1    -1 6099006 435462.6 27.21723   NA
    ## CP04 423.2363 423.2266 423.2459 -1    -1    -1 2708391 174252.7 14.74527   NA
    ## CP05 427.2681 427.2574 427.2779 -1    -1    -1 6302089 461385.6 32.50050   NA
    ## CP06 437.2375 437.2254 437.2488 -1    -1    -1 7523070 517917.6 34.37645   NA
    ##           maxf sample
    ## CP01  814693.1      1
    ## CP02  732119.9      1
    ## CP03 1018994.8      1
    ## CP04  435858.5      1
    ## CP05 1125644.3      1
    ## CP06 1282906.5      1

# 3 Calibration

The `calibrate` method can be used to correct the m/z values of identified peaks. The currently implemented method requires identified peaks and a list of m/z values for known calibrants. The identified peaks m/z values are then adjusted based on the differences between the calibrants’ m/z values and the m/z values of the closest peaks (within a user defined permitted maximal distance). Note that this method does presently only calibrate identified peaks, but not the original m/z values in the spectra.

Below we demonstrate the `calibrate` method on one of the data files with artificially defined calibration m/z values. We first subset the data set to the first data file, extract the m/z values of 3 peaks and modify the values slightly.

    ## Subset to the first file.
    first_file <- filterFile(ham_prep, file = 1)

    ## Extract 3 m/z values
    calib_mz <- chromPeaks(first_file)[c(1, 4, 7), "mz"]
    calib_mz <- calib_mz + 0.00001 * runif(1, 0, 0.4) * calib_mz + 0.0001

Next we calibrate the data set using the previously defined artificial calibrants. We are using the `"edgeshift"` method for calibration that adjusts all peaks within the range of the m/z values of the calibrants using a linear interpolation and shifts all chromatographic peaks outside of that range by a constant factor (the difference between the lowest respectively largest calibrant m/z with the closest peak’s m/z). Note that in a real use case, the m/z values would obviously represent known m/z of calibrants and would not be defined on the actual data.

    ## Set-up the parameter class for the calibration
    prm <- CalibrantMassParam(mz = calib_mz, method = "edgeshift",
                              mzabs = 0.0001, mzppm = 5)
    first_file_calibrated <- calibrate(first_file, param = prm)

To evaluate the calibration we plot below the difference between the adjusted and raw m/z values (y-axis) against the raw m/z values.

    diffs <- chromPeaks(first_file_calibrated)[, "mz"] -
        chromPeaks(first_file)[, "mz"]

    plot(x = chromPeaks(first_file)[, "mz"], xlab = expression(m/z[raw]),
         y = diffs, ylab = expression(m/z[calibrated] - m/z[raw]))

# 4 Correspondence

Correspondence aims to group peaks across samples to define the features (ions with the same m/z values). Peaks from single spectrum, direct injection MS experiments can be grouped with the MZclust method. Below we perform the correspondence analysis with the `groupChromPeaks` method using default settings.

    ## Using default settings but define sample group assignment
    mzc_prm <- MzClustParam(sampleGroups = ham_prep$sample_group)
    ham_prep <- groupChromPeaks(ham_prep, param = mzc_prm)

Getting an overview of the performed processings:

    ham_prep

    ## MSn experiment data ("XCMSnExp")
    ## Object size in memory: 0.04 Mb
    ## - - - Spectra data - - -
    ##  MS level(s): 1
    ##  Number of spectra: 4
    ##  MSn retention times: -1:59 - -1:59 minutes
    ## - - - Processing information - - -
    ## Data loaded [Thu Jun 19 22:54:55 2025]
    ##  MSnbase version: 2.35.0
    ## - - - Meta data  - - -
    ## phenoData
    ##   rowNames: 1 2 3 4
    ##   varLabels: filename sample_group
    ##   varMetadata: labelDescription
    ## Loaded from:
    ##   [1] HAM004_641fE_14-11-07--Exp1.extracted.mzML...  [4] HAM005_641fE_14-11-07--Exp2.extracted.mzML
    ##   Use 'fileNames(.)' to see all files.
    ## protocolData: none
    ## featureData
    ##   featureNames: F1.S1 F2.S1 F3.S1 F4.S1
    ##   fvarLabels: fileIdx spIdx ... spectrum (36 total)
    ##   fvarMetadata: labelDescription
    ## experimentData: use 'experimentData(object)'
    ## - - - xcms preprocessing - - -
    ## Chromatographic peak detection:
    ##  method: MSW
    ##  38 peaks identified in 4 samples.
    ##  On average 9.5 chromatographic peaks per sample.
    ## Correspondence:
    ##  method: mzClust
    ##  20 features identified.
    ##  Median mz range of features: 9.1553e-05
    ##  Median rt range of features: 0

The peak group information, i.e. the feature definitions can be accessed with the `featureDefinitions` method.

    featureDefinitions(ham_prep)

    ## DataFrame with 20 rows and 10 columns
    ##          mzmed     mzmin     mzmax     rtmed     rtmin     rtmax    npeaks
    ##      <numeric> <numeric> <numeric> <numeric> <numeric> <numeric> <numeric>
    ## FT01   402.285   402.285   402.286        -1        -1        -1         2
    ## FT02   403.237   403.237   403.237        -1        -1        -1         4
    ## FT03   405.109   405.109   405.109        -1        -1        -1         2
    ## FT04   409.184   409.184   409.185        -1        -1        -1         2
    ## FT05   410.144   410.144   410.145        -1        -1        -1         2
    ## ...        ...       ...       ...       ...       ...       ...       ...
    ## FT16   437.238   437.238   437.238        -1        -1        -1         2
    ## FT17   438.240   438.240   438.240        -1        -1        -1         2
    ## FT18   439.151   439.151   439.151        -1        -1        -1         2
    ## FT19   441.130   441.130   441.131        -1        -1        -1         2
    ## FT20   445.293   445.292   445.293        -1        -1        -1         2
    ##           ham4      ham5     peakidx
    ##      <numeric> <numeric>      <list>
    ## FT01         0         2       16,28
    ## FT02         2         2 17,29,1,...
    ## FT03         0         2       18,30
    ## FT04         2         0        10,2
    ## FT05         0         2       19,31
    ## ...        ...       ...         ...
    ## FT16         2         0        6,13
    ## FT17         2         0        7,14
    ## FT18         0         2       26,37
    ## FT19         0         2       38,27
    ## FT20         2         0        15,8

Plotting the raw data for direct injection samples involves a little more processing than for LC/GC-MS data in which we can simply use the `chromatogram` method to extract the data. Below we extract the m/z-intensity pairs for the peaks associated with the first feature. We thus first identify the peaks for that feature and define their m/z values range. Using this range we can subsequently use the `filterMz` function to sub-set the full data set to the signal associated with the feature’s peaks. On that object we can then call the `mz` and `intensity` functions to extract the data.

    ## Get the peaks belonging to the first feature
    pks <- chromPeaks(ham_prep)[featureDefinitions(ham_prep)$peakidx[[1]], ]

    ## Define the m/z range
    mzr <- c(min(pks[, "mzmin"]) - 0.001, max(pks[, "mzmax"]) + 0.001)

    ## Subset the object to the m/z range
    ham_prep_sub <- filterMz(ham_prep, mz = mzr)

    ## Extract the mz and intensity values
    mzs <- mz(ham_prep_sub, bySample = TRUE)
    ints <- intensity(ham_prep_sub, bySample = TRUE)

    ## Plot the data
    plot(3, 3, pch = NA, xlim = range(mzs), ylim = range(ints), main = "FT01",
         xlab = "m/z", ylab = "intensity")
    ## Define colors
    cols <- rep("#ff000080", length(mzs))
    cols[ham_prep_sub$sample_group == "ham5"] <- "#0000ff80"
    tmp <- mapply(mzs, ints, cols, FUN = function(x, y, col) {
        points(x, y, col = col, type = "l")
    })

To access the actual intensity values of each feature in each sample the `featureValue` method can be used. The setting `value = "into"` tells the function to return the integrated signal for each peak (one representative peak) per sample.

    feat_vals <- featureValues(ham_prep, value = "into")
    head(feat_vals)

    ##      HAM004_641fE_14-11-07--Exp1.extracted.mzML
    ## FT01                                         NA
    ## FT02                                    4735258
    ## FT03                                         NA
    ## FT04                                    4158405
    ## FT05                                         NA
    ## FT06                                    6099006
    ##      HAM004_641fE_14-11-07--Exp2.extracted.mzML
    ## FT01                                         NA
    ## FT02                                    6202418
    ## FT03                                         NA
    ## FT04                                    5004546
    ## FT05                                         NA
    ## FT06                                    4950642
    ##      HAM005_641fE_14-11-07--Exp1.extracted.mzML
    ## FT01                                    4095293
    ## FT02                                    4811391
    ## FT03                                    2982453
    ## FT04                                         NA
    ## FT05                                    2872023
    ## FT06                                         NA
    ##      HAM005_641fE_14-11-07--Exp2.extracted.mzML
    ## FT01                                    4804763
    ## FT02                                    2581183
    ## FT03                                    2268984
    ## FT04                                         NA
    ## FT05                                    2133219
    ## FT06                                         NA

`NA` is reported for features in samples for which no peak was identified at the feature’s m/z value. In some instances there might still be a signal at the feature’s position in the raw data files, but the peak detection failed to identify a peak. For these cases signal can be recovered using the `fillChromPeaks` method that integrates all raw signal at the feature’s location. If there is no signal at that location an `NA` is reported.

    ham_prep <- fillChromPeaks(ham_prep, param = FillChromPeaksParam())

    head(featureValues(ham_prep, value = "into"))

    ##      HAM004_641fE_14-11-07--Exp1.extracted.mzML
    ## FT01                                   768754.0
    ## FT02                                  4735257.5
    ## FT03                                   652566.6
    ## FT04                                  4158404.5
    ## FT05                                   652201.1
    ## FT06                                  6099006.3
    ##      HAM004_641fE_14-11-07--Exp2.extracted.mzML
    ## FT01                                  1230140.4
    ## FT02                                  6202417.6
    ## FT03                                   374109.9
    ## FT04                                  5004546.3
    ## FT05                                   403448.4
    ## FT06                                  4950641.7
    ##      HAM005_641fE_14-11-07--Exp1.extracted.mzML
    ## FT01                                    4095293
    ## FT02                                    4811391
    ## FT03                                    2982453
    ## FT04                                    1221031
    ## FT05                                    2872023
    ## FT06                                    1573988
    ##      HAM005_641fE_14-11-07--Exp2.extracted.mzML
    ## FT01                                  4804762.5
    ## FT02                                  2581183.1
    ## FT03                                  2268984.5
    ## FT04                                  1241294.4
    ## FT05                                  2133219.4
    ## FT06                                   977694.5

# 5 Further analysis

Further analysis, i.e. detection of features/metabolites with significantly different abundances, or PCA analyses can be performed on the feature matrix using functionality from other R packages, such as limma.

# 6 Session information

    sessionInfo()

    ## R version 4.5.1 (2025-06-13 ucrt)
    ## Platform: x86_64-w64-mingw32/x64
    ## Running under: Windows Server 2022 x64 (build 20348)
    ##
    ## Matrix products: default
    ##   LAPACK version 3.12.1
    ##
    ## locale:
    ## [1] LC_COLLATE=C
    ## [2] LC_CTYPE=English_United States.utf8
    ## [3] LC_MONETARY=English_United States.utf8
    ## [4] LC_NUMERIC=C
    ## [5] LC_TIME=English_United States.utf8
    ##
    ## time zone: America/New_York
    ## tzcode source: internal
    ##
    ## attached base packages:
    ## [1] stats4    stats     graphics  grDevices utils     datasets  methods
    ## [8] base
    ##
    ## other attached packages:
    ##  [1] MassSpecWavelet_1.75.1 pheatmap_1.0.13        faahKO_1.49.1
    ##  [4] MSnbase_2.35.0         ProtGenerics_1.41.0    S4Vectors_0.47.0
    ##  [7] mzR_2.43.0             Rcpp_1.0.14            Biobase_2.69.0
    ## [10] BiocGenerics_0.55.0    generics_0.1.4         MsFeatures_1.17.1
    ## [13] xcms_4.7.2             BiocParallel_1.43.4    BiocStyle_2.37.0
    ##
    ## loaded via a namespace (and not attached):
    ##  [1] DBI_1.2.3                   rlang_1.1.6
    ##  [3] magrittr_2.0.3              clue_0.3-66
    ##  [5] matrixStats_1.5.0           compiler_4.5.1
    ##  [7] vctrs_0.6.5                 reshape2_1.4.4
    ##  [9] stringr_1.5.1               pkgconfig_2.0.3
    ## [11] MetaboCoreUtils_1.17.1      crayon_1.5.3
    ## [13] fastmap_1.2.0               magick_2.8.7
    ## [15] XVector_0.49.0              rmarkdown_2.29
    ## [17] UCSC.utils_1.5.0            preprocessCore_1.71.0
    ## [19] tinytex_0.57                purrr_1.0.4
    ## [21] xfun_0.52                   MultiAssayExperiment_1.35.3
    ## [23] cachem_1.1.0                GenomeInfoDb_1.45.4
    ## [25] jsonlite_2.0.0              progress_1.2.3
    ## [27] DelayedArray_0.35.2         prettyunits_1.2.0
    ## [29] parallel_4.5.1              cluster_2.1.8.1
    ## [31] R6_2.6.1                    bslib_0.9.0
    ## [33] stringi_1.8.7               RColorBrewer_1.1-3
    ## [35] limma_3.65.1                GenomicRanges_1.61.0
    ## [37] jquerylib_0.1.4             bookdown_0.43
    ## [39] SummarizedExperiment_1.39.0 iterators_1.0.14
    ## [41] knitr_1.50                  IRanges_2.43.0
    ## [43] BiocBaseUtils_1.11.0        Matrix_1.7-3
    ## [45] igraph_2.1.4                tidyselect_1.2.1
    ## [47] dichromat_2.0-0.1           abind_1.4-8
    ## [49] yaml_2.3.10                 doParallel_1.0.17
    ## [51] codetools_0.2-20            affy_1.87.0
    ## [53] lattice_0.22-7              tibble_3.3.0
    ## [55] plyr_1.8.9                  signal_1.8-1
    ## [57] evaluate_1.0.4              Spectra_1.19.2
    ## [59] pillar_1.10.2               affyio_1.79.0
    ## [61] BiocManager_1.30.26         MatrixGenerics_1.21.0
    ## [63] foreach_1.5.2               MALDIquant_1.22.3
    ## [65] ncdf4_1.24                  hms_1.1.3
    ## [67] ggplot2_3.5.2               scales_1.4.0
    ## [69] MsExperiment_1.11.1         glue_1.8.0
    ## [71] lazyeval_0.2.2              tools_4.5.1
    ## [73] mzID_1.47.0                 data.table_1.17.6
    ## [75] QFeatures_1.19.2            vsn_3.77.0
    ## [77] fs_1.6.6                    XML_3.99-0.18
    ## [79] grid_4.5.1                  impute_1.83.0
    ## [81] tidyr_1.3.1                 MsCoreUtils_1.21.0
    ## [83] PSMatch_1.13.1              cli_3.6.5
    ## [85] S4Arrays_1.9.1              dplyr_1.1.4
    ## [87] AnnotationFilter_1.33.0     pcaMethods_2.1.0
    ## [89] gtable_0.3.6                sass_0.4.10
    ## [91] digest_0.6.37               SparseArray_1.9.0
    ## [93] farver_2.1.2                htmltools_0.5.8.1
    ## [95] lifecycle_1.0.4             httr_1.4.7
    ## [97] statmod_1.5.0               MASS_7.3-65