quantify-methods.RdThis method quantifies individual "Spectrum"
objects or full "MSnExp" experiments. Current,
MS2-level isobar tagging using iTRAQ and TMT (or any arbitrary peaks
of interest, see "ReporterIons") and MS2-level
label-free quantitation (spectral counting, spectral index or spectral
abundance factor) are available.
Isobaric tag peaks of single spectra or complete experiments can be
quantified using appropriate methods. Label-free quantitation
is available only for MSnExp experiments.
Since version 1.13.5, parallel quantitation is supported by the
BiocParallel package and controlled by the BPPARAM
argument.
An instance of class "Spectrum" (isobaric
tagging only) or "MSnExp".
Peak quantitation method. For isobaric tags, one of, possibly
abreviated "trapezoidation", "max", or
"sum". These methods return respectively the area under the
peak(s), the maximum of the peak(s) or the sum of all intensities of
the peak(s).
For label-free quantitation, one of "SI" (spectral index),
"SIgi" (global intensity spectral index), "SIn"
(normalised spectral index), "SAF" (spectral abundance
factor) or "NSAF" (normalised spectral abundance factor).
Finally, the simple "count" method counts the occurrence of
the respective spectra (at this stage all 1s) that can then be used
as input to combineFeatures to implement spectra
counting.
An instance of class "ReporterIons" that defines
the peak(s) to be quantified. For isobaric tagging only.
For isobaric tagging only. If strict is FALSE (default), the
quantitation is performed using data points along the entire width
of a peak. If strict is set to TRUE, once the apex(es) is/are
identified, only data points within apex +/- width of reporter (see
"ReporterIons") are used for quantitation.
Support for parallel processing using the BiocParallel
infrastructure. When missing (default), the default registered
BiocParallelParam parameters are applied using
bpparam(). Alternatively, one can pass a valid
BiocParallelParam parameter instance: SnowParam,
MulticoreParam, DoparParam, ... see the
BiocParallel package for details.
Deprecated. Please see BPPARAM.
Should the qual slot be populated. Default is TRUE.
A character giving the peptide sequence column in the feature
data. Default is "sequence".
Verbose of the output (only for MSnExp objects).
Further arguments passed to the quantitation functions.
"ReporterIons" define specific MZ at which peaks
are expected and a window around that MZ value. A peak of interest is
searched for in that window. Since version 1.1.2, warnings are not
thrown anymore in case no data is found in that region or if the peak
extends outside the window. This can be checked manually after
quantitation, by inspecting the quantitation data (using the
exprs accessor) for NA values or by comaring the
lowerMz and upperMz columns in the
"MSnSet" qual slot against the respective
expected mz(reporters) +/- width(reporters).
Once the range of the curve is found, quantification is performed. If
no data points are found in the expected region, NA is returned
for the reporter peak MZ.
Note that for label-free, spectra that have not been identified (the
corresponding fields in the feature data are populated with NA
values) or that have been uniquely assigned to a protein (the
nprot feature data is greater that 1) are removed prior to
quantitation. The latter does not apply for method = "count"
but can be applied manually with
removeMultipleAssignment.
signature(object = "MSnExp", method = "character", reporters
= "ReporterIons", verbose = "logical", ...)For isobaric tagging, quantifies peaks defined in reporters
using method in all spectra of the MSnExp object. If
verbose is set to TRUE, a progress bar will be displayed.
For label-free quantitation, the respective quantitation methods
and normalisations are applied to the spectra. These methods
require two additional arguments (...), namely the protein
accession of identifiers (fcol, with detault value
"DatabaseAccess") and the protein lengths (plength,
with default value "DBseqLength"). These values are
available of the identification data had been collated using
addIdentificationData.
An object of class "MSnSet" is returned
containing the quantified feature expression and all meta data
inherited from the MSnExp object argument.
signature(object = "Spectrum", method = "character",
reporters = "ReporterIons")Quantifies peaks defined in reporters using method
in the Spectrum object (isobaric tagging only).
A list of length 2 will be returned. The first element, named
peakQuant, is a 'numeric' of length equal to
length(reporters) with quantitation of the reporter peaks
using method.
The second element, names curveStats, is a 'data.frame' of
dimension length(reporters) times 7 giving, for each
reporter curve parameters: maximum intensity ('maxInt'),
number of maxima ('nMaxInt'), number of data points defined
the curve ('baseLength'), lower and upper MZ values for the
curve ('lowerMz' and 'upperMz'), reporter
('reporter') and precursor MZ value ('precursor')
when available.
For details about the spectral index (SI), see Griffin NM, Yu J, Long F, Oh P, Shore S, Li Y, Koziol JA, Schnitzer JE. Label-free, normalized quantification of complex mass spectrometry data for proteomic analysis. Nat Biotechnol. 2010 Jan;28(1):83-9. doi: 10.1038/nbt.1592. PMID: 20010810; PubMed Central PMCID: PMC2805705.
For details about the spectra abundance factor, see Paoletti AC, Parmely TJ, Tomomori-Sato C, Sato S, Zhu D, Conaway RC, Conaway JW, Florens L, Washburn MP. Quantitative proteomic analysis of distinct mammalian Mediator complexes using normalized spectral abundance factors. PNAS. 2006 Dec 12;103(50):18928-33. PMID: 17138671; PubMed Central PMCID: PMC1672612.
## Quantifying a full experiment using iTRAQ4-plex tagging
data(itraqdata)
msnset <- quantify(itraqdata, method = "trap", reporters = iTRAQ4)
msnset
#> MSnSet (storageMode: lockedEnvironment)
#> assayData: 55 features, 4 samples
#> element names: exprs
#> protocolData: none
#> phenoData
#> sampleNames: iTRAQ4.114 iTRAQ4.115 iTRAQ4.116 iTRAQ4.117
#> varLabels: mz reporters
#> varMetadata: labelDescription
#> featureData
#> featureNames: X1 X10 ... X9 (55 total)
#> fvarLabels: spectrum ProteinAccession ... collision.energy (15 total)
#> fvarMetadata: labelDescription
#> experimentData: use 'experimentData(object)'
#> Annotation: No annotation
#> - - - Processing information - - -
#> Data loaded: Wed May 11 18:54:39 2011
#> Updated from version 0.3.0 to 0.3.1 [Fri Jul 8 20:23:25 2016]
#> iTRAQ4 quantification by trapezoidation: Thu Mar 14 06:11:31 2024
#> MSnbase version: 1.1.22
## specifying a custom parallel framework
## bp <- MulticoreParam(2L) # on Linux/OSX
## bp <- SnowParam(2L) # on Windows
## quantify(itraqdata[1:10], method = "trap", iTRAQ4, BPPARAM = bp)
## Checking for non-quantified peaks
sum(is.na(exprs(msnset)))
#> [1] 1
## Quantifying a single spectrum
qty <- quantify(itraqdata[[1]], method = "trap", iTRAQ4[1])
qty$peakQuant
#> iTRAQ4.114
#> 1347.616
qty$curveStats
#> maxInt nMaxInt baseLength lowerMz upperMz reporter precursor
#> [1,] 197455.8 1 11 114.1012 114.1196 114.1112 520.7833
## Label-free quantitation
## Raw (mzXML) and identification (mzid) files
quantFile <- dir(system.file(package = "MSnbase", dir = "extdata"),
full.name = TRUE, pattern = "mzXML$")
identFile <- dir(system.file(package = "MSnbase", dir = "extdata"),
full.name = TRUE, pattern = "dummyiTRAQ.mzid")
msexp <- readMSData(quantFile)
msexp <- addIdentificationData(msexp, identFile)
fData(msexp)$DatabaseAccess
#> [1] "ECA0984" "ECA1028" NA NA "ECA0510"
si <- quantify(msexp, method = "SIn")
processingData(si)
#> - - - Processing information - - -
#> Data loaded: Thu Mar 14 06:11:32 2024
#> Filtered 2 unidentified peptides out [Thu Mar 14 06:11:32 2024]
#> Quantitation by total ion current [Thu Mar 14 06:11:32 2024]
#> Combined 3 features into 3 using sum: Thu Mar 14 06:11:32 2024
#> Quantification by SIn [Thu Mar 14 06:11:32 2024]
#> MSnbase version: 2.29.4
exprs(si)
#> dummyiTRAQ.mzXML
#> ECA0510 0.0006553518
#> ECA0984 0.0035384487
#> ECA1028 0.0002684726
saf <- quantify(msexp, method = "NSAF")
processingData(saf)
#> - - - Processing information - - -
#> Data loaded: Thu Mar 14 06:11:32 2024
#> Filtered 2 unidentified peptides out [Thu Mar 14 06:11:32 2024]
#> Filtered 0 unidentified peptides out [Thu Mar 14 06:11:32 2024]
#> Quantitation by count [Thu Mar 14 06:11:32 2024]
#> Combined 3 features into 3 using user-defined function: Thu Mar 14 06:11:32 2024
#> Quantification by NSAF [Thu Mar 14 06:11:32 2024]
#> MSnbase version: 2.29.4
exprs(saf)
#> dummyiTRAQ.mzXML
#> ECA0510 0.4306167
#> ECA0984 0.3094475
#> ECA1028 0.2599359