# Processing Tandem-MS and MSn data with xcms

S. Neumann and K. Kutzera

#### 9 October 2019

#### Package

xcms 3.6.2

# 1 Introduction

This document describes how to use `xcms` for processing of Tandem-MS and MS\(^{n}\) data from e.g. triple-quad, QTOF, ion trap or orbitrap mass spectrometers. This uses still the old user interface.

    library(xcms)
    library(msdata)

    ## Disable parallel processing
    register(SerialParam())

# 2 Raw data file preparation

The `xcms` package reads full-scan LC/MS data and associated Tandem-MS and MS\(^{n}\) spectra from mzData and mzXML files (CDF does not support MS\(^{n}\)).

For the purposes of demonstration, we will use a some sample files included in the `msdata` package. The (truncated) raw data files are contained in the data directory. To access the mzData files, we first locate the data directory in the `msdata` package.

    mzdatapath <- system.file("iontrap", package = "msdata")
    list.files(mzdatapath, recursive = TRUE)

    ## [1] "DhexD4_1.mzData"  "DhexD4_2.mzData"  "DhexD4_3.mzData"
    ## [4] "extracted.mzData"

# 3 Accessing, combining and visualising MSn spectra

The MS\(^{n}\) spectra are read into xcms just like plain LC-MS files. Inclusion of the additional scans has to be explicitely enabled. The xcmsRaw summary shows the additional content:

    library(xcms)
    mzdatafiles <- list.files(mzdatapath, pattern = "extracted.mzData",
                              recursive = TRUE, full.names = TRUE)
    xraw <- xcmsRaw(mzdatafiles[1], includeMSn=TRUE)

    ## Create profile matrix with method 'bin' and step 1 ... OK

    xraw

    ## An "xcmsRaw" object with 100 mass spectra
    ##
    ## Time range: 1190-1309.2 seconds (19.8-21.8 minutes)
    ## Mass range: 500-849.9333 m/z
    ## Intensity range: 100008-828487000
    ##
    ## MSn data on  22  mass(es)
    ##  with  44  MSn spectra
    ## Profile method: bin
    ## Profile step: 1 m/z (351 grid points from 500 to 850 m/z)
    ##
    ## Memory usage: 2.71 MB

There is also a new peak picker, which actually returns all parent ions as annotated in the mzData / mzXML files. A warning will be issued if this is not present, either because of a poor converter or if `xraw` is read from a CDF file:

    peaks <- findPeaks(xraw, method="MS1")

By giving larger windows, subsets of spectra can be returned, and these can be combined to obtain so called compound spectra containing e.g. MS\(^{2}\) to MS\(^{5}\).

# 4 xcmsFragments

A xcmsFragments-Object contains a Table with informations about all Peaks from MS1 to MS\(^{n}\) of one Experiment, including mz-value, retentiontime, MSlevel, and parentpeak. The data can be used for visualization or (later) computing alignments between different Experiments. The object is created as empty and can be filled with `collect()`. This method requires a peaktable or a xcmsSet for the MS1peaks and the original xcmsRaw for the msn-Data.

    xs <- xcmsSet(mzdatafiles, method = "MS1")

    xfrag <- xcmsFragments(xs)

    ## Create profile matrix with method 'bin' and step 1 ... OK

    ## 394 Peaks picked, 0 MSn-Specs ignored.

    xfrag

    ## An "xcmsFragments" object with  394  peaks in 77 Spectra
    ## From Level 1 to 3 Number of Samples:  1 .
    ##
    ## Sample 1 :
    ##     37 Peaks in Level 1
    ##     351 Peaks in Level 2
    ##     6 Peaks in Level 3
    ##
    ## Memory usage: 0.029 MB

plotTree prints out a simplyfied tree of the peaks in a `xcmsFragments`. The user can define borders for the retentionTime and for the MassRange, he can also print the tree for a single MS1-Peak. PlotTree uses the package `RgraphViz` and plots the tree text-based if requested or if the package cannot be found.

Below we simply print the textual representation of the fragment tree.

    plotTree(xfrag, xcmsFragmentPeakID = 6, textOnly = TRUE)

    ## |##6_M814T1205.2F814
    ## |     ##75_M814T1206.52F673.2994
    ## |          ##85_M814T1207.13F586.9333
    ## |          ##86_M814T1207.13F588.1333
    ## |     ##76_M814T1206.52F672.0354
    ## |     ##77_M814T1206.52F573.2417
    ## |     ##78_M814T1206.52F658.2642
    ## |     ##79_M814T1206.52F742.0226
    ## |     ##80_M814T1206.52F742.4218
    ## |     ##81_M814T1206.52F657.1997
    ## |     ##82_M814T1206.52F587.0795
    ## |     ##83_M814T1206.52F755.1951
    ## |     ##84_M814T1206.52F743.2867