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Information flow in biological systems (Source Central dogma of biology on Wikipedia).
There are three main biological entities that respectively store information, act as data intermediates, and the functional units, and the figure below show how information flows between these three levels.
DNA, that lives in the nucleus of cells, is the central information storage mechanism, and encodes the blueprint of the functional units as genes. DNA is transcribed into messenger RNA (mRNA), that re-localises outside the nucleus and is further processed into its mature exon-only form after removal of the non-coding introns sequences. Finally, the mRNA is translated by the ribosomial machinery into proteins directly into the endoplasmic reticulum (ER) where they are then redirected to their final destination.
In addition to the standard information worflow where DNA is transcribed into RNA that itself is translated into proteins, there is also reverse transcription, that generates (complementary) DNA from and RNA molecule, as well as replication of DNA (during cell division) and RNA molecules.
Proteins as the functional units in all living organisms, and they are highly dynamic. The caterpillar and the resulting butterfly have the same genome. The complement of all the expressed proteins, termed the proteome is however very different.
There are different modalities of the proteome that are of interest. In addition to the presence or amount of protein in a biological samples, it is also important to study the interactions between proteins forming protein-protein complexes, the presence of post-transcriptional modification (such as, for example, phosphorylations), the rate at which proteins are produced and degraded, or where the proteins reside inside a cell.
The technique of choice to study proteins in a high throughput way is mass spectrometry.
A beginner’s guide to mass spectrometry–based proteomics (Sinha and Mann 2020) is an approachable introduction to sample preparation, mass spectrometry and data analysis.
The MSnbase
package.
We are going to use the Bioconductor (Huber et al. 2015) MSnbase
package
(Gatto and Lilley 2012; Gatto, Gibb, and Rainer 2020), which can be install with the BiocManager
package, available from CRAN. If BiocManager
isn’t available on your
computer, install it with:
install.packages("BiocManager")
Now, install MSnbase
and its dependencies with
BiocManager::install("MSnbase")
For additional information on how to analyse mass spectrometry-based proteomics data, refer to (Gatto and Christoforou 2014) and (Gatto 2019), or explore the the proteomics- and mass spectrometry-related packages on the Bioconductor page
The recent R for Mass Spectrometry initiative, and the documetation book provide further details and state-of-the-art infrastructure for MS and proteomics data analysis.
The R for Mass Spectrometry intiative.
Mass spectrometry (MS) is a technology that separates charged molecules (ions) based on their mass to charge ratio (M/Z). It is often coupled to chromatography (liquid LC, but can also be gas-based GC). The time an analytes takes to elute from the chromatography column is the retention time.
An mass spectrometer is composed of three components:
When using mass spectrometry for proteomics, the proteins are first digested with a protease such as trypsin. In mass shotgun proteomics, the analytes assayed in the mass spectrometer are peptides.
Often, ions are subjected to more than a single MS round. After a first round of separation, the peaks in the spectra, called MS1 spectra, represent peptides. At this stage, the only information we possess about these peptides are their retention time and their mass-to-charge (we can also infer their charge be inspecting their isotopic envelope, i.e the peaks of the individual isotopes, see below), which is not enough to infer their identify (i.e. their sequence).
In MSMS (or MS2), the settings of the mass spectrometer are set automatically to select a certain number of MS1 peaks (for example 20). Once a narrow M/Z range has been selected (corresponding to one high-intensity peak, a peptide, and some background noise), it is fragmented (using for example collision-induced dissociation (CID), higher energy collisional dissociation (HCD) or electron-transfer dissociation (ETD)). The fragment ions are then themselves separated in the analyser to produce a MS2 spectrum. The unique fragment ion pattern can then be used to infer the peptide sequence using de novo sequencing (when the spectrum is of high enough quality) of using a search engine such as, for example Mascot, MSGF+, …, that will match the observed, experimental spectrum to theoratical spectra (see details below).
The animation below show how 25 ions different ions (i.e. having different M/Z values) are separated throughout the MS analysis and are eventually detected (i.e. quantified). The final frame shows the hypothetical spectrum.
The figures below illustrate the two rounds of MS. The spectrum on the left is an MS1 spectrum acquired after 21 minutes and 3 seconds of elution. 10 peaks, highlited by dotted vertical lines, were selected for MS2 analysis. The peak at M/Z 460.79 (488.8) is highlighted by a red (orange) vertical line on the MS1 spectrum and the fragment spectra are shown on the MS2 spectrum on the top (bottom) right figure.
The figures below represent the 3 dimensions of MS data: a set of spectra (M/Z and intensity) of retention time, as well as the interleaved nature of MS1 and MS2 (and there could be more levels) data.
Let’s read a very small raw MS data file into R using the readMSData
from the MSnbase
package. The file that we are going to load is also
available in the package.
MSnbase
packagelibrary("MSnbase")
dummyiTRAQ.mzXML
filerawf <- dir(system.file(package = "MSnbase", dir = "extdata"),
full.name = TRUE,
pattern = "mzXML$")
basename(rawf)
## [1] "dummyiTRAQ.mzXML"
readMSData
function.x <- readMSData(rawf)
x
## MSn experiment data ("MSnExp")
## Object size in memory: 0.18 Mb
## - - - Spectra data - - -
## MS level(s): 2
## Number of spectra: 5
## MSn retention times: 25:01 - 25:02 minutes
## - - - Processing information - - -
## Data loaded: Mon Apr 29 12:11:17 2024
## MSnbase version: 2.28.1
## - - - Meta data - - -
## phenoData
## rowNames: dummyiTRAQ.mzXML
## varLabels: sampleNames
## varMetadata: labelDescription
## Loaded from:
## dummyiTRAQ.mzXML
## protocolData: none
## featureData
## featureNames: F1.S1 F1.S2 ... F1.S5 (5 total)
## fvarLabels: spectrum
## fvarMetadata: labelDescription
## experimentData: use 'experimentData(object)'
The object that is returned by readMSData
is of class MSnExp
, that
can store, access and manipulate raw MS data. Note that here we are
focusing on MS-based proteomics data, but this also applied to
MS-based metabolomics data.
class(x)
## [1] "MSnExp"
## attr(,"package")
## [1] "MSnbase"
length
. Full MS acquisitions would contain hundreds
of thousands spectra.length(x)
## [1] 5
msLevel(x)
## F1.S1 F1.S2 F1.S3 F1.S4 F1.S5
## 2 2 2 2 2
rtime(x)
## F1.S1 F1.S2 F1.S3 F1.S4 F1.S5
## 1501.35 1501.59 1501.85 1502.07 1502.31
precursorMz(x)
## F1.S1 F1.S2 F1.S3 F1.S4 F1.S5
## 645.3741 546.9586 645.3741 716.3405 437.8040
precursorIntensity(x)
## F1.S1 F1.S2 F1.S3 F1.S4 F1.S5
## 47659400 26356100 23432400 24854800 7052960
[[
and plot them.x[[3]]
## Object of class "Spectrum2"
## Precursor: 645.3741
## Retention time: 25:02
## Charge: 2
## MSn level: 2
## Peaks count: 2125
## Total ion count: 150838188
plot(x[[3]])
For the rest of this tutorial, we will be using a
slightly larger dataset (still tiny compared to full acquisitions)
that is distributed with the MSnbase
package. Load it as shown below
and compute the number of spectra available in that dataset, their MS
level, and the retention time range over which these spectra have been
acquired.
data(itraqdata)
length(itraqdata)
## [1] 55
unique(msLevel(itraqdata))
## [1] 2
formatRt(range(rtime(itraqdata)))
## [1] "19:09" "50:18"
This object also contains additional metadata for each spectrum, that
can be accessed, as a data.frame
, with fData
.
The raw data is still a long way of obtaining biologically relevant proteomics data. The first step to obtain proteomics data is to identify the peptides that have been acquired in the MS. Peptide identification work by comparing expected and observed spectra. As shown below, when a precursor peptide ion is fragmented in a CID cell, it breaks at specific bonds, producing sets of peaks (a, b, c and x, y, z) that can be predicted.
It is thus possible to calculate the expected set of fagment peaks for a given peptide, such as SIGFEGDSIGR below.
calculateFragments("SIGFEGDSIGR")
## mz ion type pos z seq
## 1 88.03931 b1 b 1 1 S
## 2 201.12337 b2 b 2 1 SI
## 3 258.14483 b3 b 3 1 SIG
## 4 405.21324 b4 b 4 1 SIGF
## 5 534.25583 b5 b 5 1 SIGFE
## 6 591.27729 b6 b 6 1 SIGFEG
## 7 706.30423 b7 b 7 1 SIGFEGD
## 8 793.33626 b8 b 8 1 SIGFEGDS
## 9 906.42032 b9 b 9 1 SIGFEGDSI
## 10 963.44178 b10 b 10 1 SIGFEGDSIG
## 11 175.11895 y1 y 1 1 R
## 12 232.14041 y2 y 2 1 GR
## 13 345.22447 y3 y 3 1 IGR
## 14 432.25650 y4 y 4 1 SIGR
## 15 547.28344 y5 y 5 1 DSIGR
## 16 604.30490 y6 y 6 1 GDSIGR
## [ reached 'max' / getOption("max.print") -- omitted 16 rows ]
The last step is to compare obseved and expected peaks. If there is a good match, the MS2 spectrum is assigned the peptide sequence.
itraqdata2 <- pickPeaks(itraqdata, verbose = FALSE)
s <- "SIGFEGDSIGR"
plot(itraqdata2[[14]], s, main = s)
It is also possible to plot 2 spectra to compare them directly.
plot(itraqdata2[[25]], itraqdata2[[28]],
sequences = rep("IMIDLDGTENK", 2))
In a full experiment, all possible peptides from the known (or relevant) proteome of interest (such as databases that can be downloaded from the UniProt site1 The Universal Protein Resource (UniProt) is a freely and accessible comprehensive resource for protein sequence and annotation data.) are compared to the millions of observed spectra.
Statistical challenge The matching between millions of observed and possible spectra causes real challenges due to the large search space and the risk of false positives. See (Käll et al. 2008) for further reading.
From the list of identified peptides, it is then necessary to infer the most provable proteins that were present in the biological sample.
Statistical challenge Protein inference is a difficult task, as peptides often match multiple proteins (either different isoforms or proteins stemming from different gene but with identical domains), which leads to the definition of protein groups, i.e. sets of proteins that can’t be distinguished with the set of identified peptides at hand. See (Nesvizhskii and Aebersold 2005) for further reading.
Plot the 44th spectrum of the itraqdata2
experiment. The sequence
can be accessed in the feature metadata with
fData(itraqdata2)$PeptideSequence[[44]]
## [1] LDVLDGLK
## 47 Levels: AADALLLK AAGHDGK AAKPEAPAASPAPALGAR AALESAVK ... VWVVEGSK
plot(itraqdata2[[44]], s,
main = fData(itraqdata2)$PeptideSequence[[44]])
The last step of MS data processing is to quantify peptide abundances in the biological samples. The table below summarises the different possibilites depending whether the proteins or peptides are labelled, and whether the quantitation is performed in MS1 or MS2.
Label-free | Labelled | |
---|---|---|
MS1 | XIC | SILAC, 15N |
MS2 | Counting | iTRAQ, TMT |
In spectral counting, on simply counts the number of quantified peptides that are assigned to a protein.
Isobaric tagging refers to the labelling using isobaric tags, i.e. chemical tags that have the same mass and hence can’t be distinguish by the spectrometer. The peptides of different samples (4, 6, 10 or 11) are labelled with different tags and combined prior to mass spectrometry acquisition. Given that they are isobaric, all identical peptides, irrespective of the tag and this the sample of origin, are co-analysed, up to fragmentation prior to MS2 analysis. During fragmentation, the isobaric tags fall of, fragment themselves, and result in a set of sample specific peaks. These specific peaks can be used to infer sample-specific quantitation, while the rest of the MS2 spectrum is used for identification.
In label-free quantitation, the precursor peaks that match an identified peptide are integrated of retention time and the area under that extracted ion chromatogram is used to quantify that peptide in that sample.
Figure: credit Johannes Rainer.
In SILAc quantitation, sample are grown in a medium that contains heavy amino acids (typically arginine and lysine). All proteins gown in this heavy growth medium contain the heavy form of these amino acids. Two samples, one grown in heavy medium, and one grown in normal (light) medium are then combined and analysed together. The heavy peptides precursor peaks are systematically shifted compared to the light ones, and the ratio between the height of a heavy and light peaks can be used to calculate peptide and protein fold-changes.
Figure: credit Wikimedia Commons.
Statistical challenge These different quantitation techniques come with their respective and distinct challenges, such as large quantities of raw data processing, data transformation and normalisation, and different underlying statistical models for the quantitative data (count data for spectral counting, continuous data for the others).
As its name implies, the itraqdata
is an iTRAQ-based isobar
quantitation experiment. We can visualise the reporter peaks as
follows:
plot(itraqdata[[14]], reporters = iTRAQ4, full = TRUE)
Interpret the figure above.
From the closed-up panel, we see that the 4 reporter ions indicate that there was slightly less of that peptide in the two first samples, i.e. those labelled with the 114 and 115 reporters.
The rest of the spectrum (i.e m/z > 120) correspond to the dissociated fragment ions, used for identification.
We can quantify these four peaks with the quantify
method, to
produce and object of class MSnSet
containing quantitation data. The
quantitation values can be accessed with exprs
. This data also
contains feature metadata that can be accessed with the fData
function.
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: Mon Apr 29 12:11:19 2024
## MSnbase version: 1.1.22
head(exprs(msnset))
## iTRAQ4.114 iTRAQ4.115 iTRAQ4.116 iTRAQ4.117
## X1 1347.6158 2247.3097 3927.6931 7661.1463
## X10 739.9861 799.3501 712.5983 940.6793
## X11 27638.3582 33394.0252 32104.2879 26628.7278
## X12 31892.8928 33634.6980 37674.7272 37227.7119
## X13 26143.7542 29677.4781 29089.0593 27902.5608
## X14 6448.0829 6234.1957 6902.8903 6437.2303
head(fData(msnset))
## spectrum ProteinAccession ProteinDescription
## X1 1 BSA bovine serum albumin
## X10 10 ECA1422 glucose-1-phosphate cytidylyltransferase
## X11 11 ECA4030 50S ribosomal subunit protein L4
## X12 12 ECA3882 chaperone protein DnaK
## X13 13 ECA1364 succinyl-CoA synthetase alpha chain
## X14 14 ECA0871 NADP-dependent malic enzyme
## PeptideSequence fileIdx retention.time precursor.mz precursor.intensity
## X1 NYQEAK 1 1149.31 520.7833 3449020
## X10 VTLVDTGEHSMTGGR 1 1503.03 573.9539 7849420
## X11 SPIWR 1 1663.61 401.7392 41253600
## X12 TAIDDALK 1 1663.86 567.8339 23549500
## X13 SILINK 1 1664.08 488.3269 13025200
## X14 DFEVVNNESDPR 1 1664.32 782.8715 18405000
## charge peaks.count tic ionCount ms.level acquisition.number
## X1 2 1922 26413754 26413754 2 2
## X10 3 1376 24482281 24482281 2 11
## X11 2 1571 231075934 231075934 2 12
## X12 2 2397 247323187 247323187 2 13
## X13 2 2574 207247502 207247502 2 14
## X14 2 1829 115317275 115317275 2 15
## collision.energy
## X1 40
## X10 40
## X11 40
## X12 40
## X13 40
## X14 40
Statistical challenge Quantitative data processing come with numerous statistical challenges such as missing data handling, aggregation of quantitation data, data normalisation, … and the effect of these on the downstream statistical tests.
In our examples, we not have processing data for the 55 peptides and 4 samples. In this data, there is only 1 missing value, corresponding to an absent reporter peak. We are going to simply drop that feature.
table(is.na(exprs(msnset)))
##
## FALSE TRUE
## 219 1
msnset <- filterNA(msnset)
In MS1 label-free experiments, given that each sample is acquired independently, the proportion of missing values can be as high several tens of percent. In such situations, removing rows with missing values isn’t possible at all. Imputation is possible, albeit tricky, as different mechanisms can be responsible for missing value that appear either at random or not at random (Lazar et al. 2016).
Next, we aggregate the spectrum-level quantitation values into
protein-level data using the median and the combineFeatures
function:
prots <- combineFeatures(msnset, fcol = "ProteinAccession",
method = "median")
head(exprs(prots))
## iTRAQ4.114 iTRAQ4.115 iTRAQ4.116 iTRAQ4.117
## BSA 1347.616 2247.310 3927.693 7661.1463
## ECA0172 17593.548 18545.620 19361.837 18328.2365
## ECA0435 4923.628 5557.818 5775.203 5079.2952
## ECA0452 1524.148 1399.897 1547.218 1563.2299
## ECA0469 1069.945 1035.689 1029.420 999.6957
## ECA0621 1101.062 1124.167 1140.093 1191.8055
Following on from here, many data processing such as normalisation, non-specific filtering, and hypothesis testing is very similar to other omics data.
The pRoloc
package.
In this section, I illustrate a use case of mass spectrometry-based
proteomics to infer sub-cellular protein localisation and the
application of statistical learning. This section requires the
pRoloc
and pRolocdata
packages (Gatto et al. 2014; Breckels et al. 2016). Both
packages can be installed with the BiocManager::install
function, as
illustrated above.
library("pRoloc")
library("pRolocdata")
The hyperLOPIT2015
data contains localisation data for 5032 proteins
in mouse embryonic stem cells (Christoforou et al. 2016). The protein
information is collected along a 20 fraction density gradient - our
data matrix has dimensions 5032 rows and 20 columns. We use PCA to
easily visualise it in 2 dimensions.
data(hyperLOPIT2015)
## set colours
setStockcol(paste0(getStockcol(), 80))
## produce PCA plot
plot2D(hyperLOPIT2015)
Each dot on the PCA plot represents a single protein. The protein is either of unknown localisation, and represented by a grey circle, or is of known localisation (and called an organelle marker) and is coloured according to its expected sub-cellular localisation.
In the next figure, we have trained an support vector machine (SVM) model and classified proteins of unknown localisation using an SVM model. The size of the dots are scaled according to the classifier score and only assignments corresponding to a false discovery rate of 5% have been considered.
sz <- exp(fData(hyperLOPIT2015)$svm.score) - 1
plot2D(hyperLOPIT2015, fcol = "final.assignment", cex = sz)
## R version 4.3.3 (2024-02-29)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 22.04.4 LTS
##
## Matrix products: default
## BLAS: /usr/lib/x86_64-linux-gnu/blas/libblas.so.3.10.0
## LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.10.0
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## [11] LC_MEASUREMENT=nl_BE.UTF-8 LC_IDENTIFICATION=C
##
## time zone: Europe/Brussels
## tzcode source: system (glibc)
##
## attached base packages:
## [1] stats4 stats graphics grDevices datasets utils methods
## [8] base
##
## other attached packages:
## [1] pRolocdata_1.40.0 pRoloc_1.42.0 BiocParallel_1.36.0
## [4] MLInterfaces_1.82.0 cluster_2.1.6 annotate_1.80.0
## [7] XML_3.99-0.16.1 AnnotationDbi_1.64.1 IRanges_2.36.0
## [10] MSnbase_2.28.1 ProtGenerics_1.34.0 S4Vectors_0.40.2
## [13] mzR_2.37.2 Rcpp_1.0.12 Biobase_2.62.0
## [16] BiocGenerics_0.48.1
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## [1] splines_4.3.3 bitops_1.0-7 filelock_1.0.3
## [4] tibble_3.2.1 hardhat_1.3.1 preprocessCore_1.64.0
## [7] pROC_1.18.5 rpart_4.1.23 lifecycle_1.0.4
## [10] doParallel_1.0.17 globals_0.16.3 lattice_0.22-6
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Gatto, Laurent, Sebastian Gibb, and Johannes Rainer. 2020. “MSnbase, Efficient and Elegant R-Based Processing and Visualisation of Raw Mass Spectrometry Data.” bioRxiv. https://doi.org/10.1101/2020.04.29.067868.
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Gatto, L, and A Christoforou. 2014. “Using R and Bioconductor for Proteomics Data Analysis.” Biochim. Biophys. Acta 1844 (1 Pt A): 42–51.
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Käll, L, J D Storey, M J MacCoss, and W S Noble. 2008. “Posterior Error Probabilities and False Discovery Rates: Two Sides of the Same Coin.” J Proteome Res 7 (1): 40–44. https://doi.org/10.1021/pr700739d.
Lazar, C, L Gatto, M Ferro, C Bruley, and T Burger. 2016. “Accounting for the Multiple Natures of Missing Values in Label-Free Quantitative Proteomics Data Sets to Compare Imputation Strategies.” J Proteome Res 15 (4): 1116–25. https://doi.org/10.1021/acs.jproteome.5b00981.
Nesvizhskii, A I, and R Aebersold. 2005. “Interpretation of Shotgun Proteomic Data: The Protein Inference Problem.” Mol Cell Proteomics 4 (10): 1419–40. https://doi.org/10.1074/mcp.R500012-MCP200.
Sinha, Ankit, and Matthias Mann. 2020. “A beginner’s guide to mass spectrometry–based proteomics.” The Biochemist, September. https://doi.org/10.1042/BIO20200057.