Grouping FTICR-MS data with xcms
In xcms: LC-MS and GC-MS Data Analysis

BiocStyle::markdown()

Introduction

library(BiocStyle)

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

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. r Biocpkg("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(xcms)
library(MassSpecWavelet)

register(SerialParam())

In this documentation we use an example data set from the r Biocpkg("msdata") package. Assuming that r Biocpkg("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.

mzdata_path <- system.file("fticr", package = "msdata")
mzdata_files <- list.files(mzdata_path, recursive = TRUE, full.names = TRUE)

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

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

## Load the data.
ham_raw <- readMSData(files = mzdata_files,
                      pdata = new("NAnnotatedDataFrame", 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)

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))

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]))

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

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

featureDefinitions(ham_prep)

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)

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"))

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 r Biocpkg("limma").