Pulsed-SILAC data analysis
2020-04-28
Abstract
This document contains a tutorial on how to perform basic pulsed SILAC data analysis using the pulsedSilac package. This includes data organization, filtering, modelling and plotting.Contents
1 Installation
To install this package, start R (version “3.6”) and enter:
if (!requireNamespace("BiocManager", quietly = TRUE))
install.packages("BiocManager")
BiocManager::install("pulsedSilac")To install the development version of the package, please do it from the development branch in github.
BiocManager::install('marcpaga/pulsedSilac')2 Introduction
SILAC (Stable Isotope Label by Amino acids in Cell culture) is a method that is often used to differentially compare protein expression between samples. Pulsed SILAC is a variant that is used to measure protein turnover. Rather than comparing samples that are labeled to completion with either heavy or light isotopes of amino acids, in pulsed SILAC cells are harvested at a series of time points after the growth media containing light amino acids was switched to media containing heavy amino acids. Each sample is then processed and analyzed by mass spectrometry. The isotope ratio each peptide at each time point can now be used to calculate the turnover rate of each measured protein in a proteome-wide fashion (Pratt et al. (2002), Doherty et al. (2005), Schwanhäusser et al. (2009)).
Figure 1: Pulsed SILAC overview
This package contains a set of functions to analyze pulsed SILAC quantitative data. Functions are provided to organize the data, calculate isotope ratios, isotope fractions, model protein turnover, compare turnover models, estimate cell growth and estimate isotope recycling. Several visualization tools are also included to perform basic data exploration, quality control, condition comparison, individual model inspection and model comparison.
3 Data organization
Many Bioconductor packages use SummarizedExperiment objects or derivatives from it to organize omics data. This is a rectangular class of object that contains assays (matrix-like organized data) in which rows represent features (genes, proteins, metabolites, …) and columns represent samples. If you are unfamiliar with this format, please read this introductory vignette.
This package uses two SummarizedExperiment derived classes: SilacProteinExperiment and SilacPeptideExperiment. It also introduces a new class, the SilacProteomicsExperiment, which encapsulates both the previous classes.
3.1 SilacProteinExperiment and SilacPeptideExperiment
The SilacProteinExperiment and SilacPeptideExperiment are, as their name indicates, dedicated to store either protein or peptide related data. These two objects are almost identical to a SummarizedExperiment object. Their only difference is the initialization of the metadata slot with what will be called metaoptions. These metaoptions contain values that are indicative of where certain information, required for the analysis, is located in the object. For example, the conditionCol value is used to indicate which column of colData has the information about the different experiment conditions.
Figure 2: SilacProteinExperiment/SilacPeptideExperiment object structure
3.1.1 Object construction
Constructing a SilacProteinExperiment or a SilacPeptideExperiment is extremely similar to constructing a SummarizedExperiment. The only additional arguments are the different metaoptions values.
Here is an example of constructing a SilacProteinExperiment:
require(pulsedSilac)
## assays
assays_protein <- list(expression = matrix(1:9, ncol = 3))
## colData
colData <- data.frame(sample = c('A1', 'A2', 'A3'),
condition = c('A', 'A', 'A'),
time = c(1, 2, 3))
## rowData
rowData_protein <- data.frame(prot_id = LETTERS[1:3])
## construct the SilacProteinExperiment
protExp <- SilacProteinExperiment(assays = assays_protein,
rowData = rowData_protein,
colData = colData,
conditionCol = 'condition',
timeCol = 'time')
protExp## class: SilacProteinExperiment
## dim: 3 3
## metadata(2): conditionCol timeCol
## assays(1): expression
## rownames: NULL
## rowData names(1): prot_id
## colnames: NULL
## colData names(3): sample condition timeHere is an example of constructing a SilacPeptideExperiment:
## assays
assays_peptide <- list(expression = matrix(1:15, ncol = 3))
## colData
colData <- data.frame(sample = c('A1', 'A2', 'A3'),
condition = c('A', 'A', 'A'),
time = c(1, 2, 3))
## rowData
rowData_peptide <- data.frame(pept_id = letters[1:5],
prot_id = c('A', 'A', 'B', 'C', 'C'))
## construct the SilacProteinExperiment
peptExp <- SilacPeptideExperiment(assays = assays_peptide,
rowData = rowData_peptide,
colData = colData,
conditionCol = 'condition',
timeCol = 'time')
peptExp## class: SilacPeptideExperiment
## dim: 5 3
## metadata(3): conditionCol timeCol proteinCol
## assays(1): expression
## rownames: NULL
## rowData names(2): pept_id prot_id
## colnames: NULL
## colData names(3): sample condition time3.1.2 Accessor functions
The accessor functions are exactly the same as in a SummarizedExperiment object: rowData(), colData(), assays() and metadata(). To access the metaoptions slot the both the metaoptions() and metadata() functions can be used.
## assays
assays(protExp)## List of length 1
## names(1): expressionassays(peptExp)## List of length 1
## names(1): expression## rowData
rowData(protExp)## DataFrame with 3 rows and 1 column
## prot_id
## <character>
## 1 A
## 2 B
## 3 CrowData(peptExp)## DataFrame with 5 rows and 2 columns
## pept_id prot_id
## <character> <character>
## 1 a A
## 2 b A
## 3 c B
## 4 d C
## 5 e C## colData
colData(protExp)## DataFrame with 3 rows and 3 columns
## sample condition time
## <character> <character> <numeric>
## 1 A1 A 1
## 2 A2 A 2
## 3 A3 A 3colData(peptExp)## DataFrame with 3 rows and 3 columns
## sample condition time
## <character> <character> <numeric>
## 1 A1 A 1
## 2 A2 A 2
## 3 A3 A 3## metaoptions
metaoptions(protExp)## $conditionCol
## [1] "condition"
##
## $timeCol
## [1] "time"metaoptions(peptExp)[['proteinCol']] <- 'prot_id'
metaoptions(peptExp)## $conditionCol
## [1] "condition"
##
## $timeCol
## [1] "time"
##
## $proteinCol
## [1] "prot_id"3.1.3 Subsetting and aggregation
Similar to the accessor function, most of the SummarizedExperiment operations, such as subsetting and aggregating, can be applied to the two classes. For subsetting these are mainly: [, subset, $; and for aggregation these are mainly: cbind, rbind and merge. For a more detailed explanation please refer to the manual.
## subsetting by rows and columns
protExp[1, 1:2]## class: SilacProteinExperiment
## dim: 1 2
## metadata(2): conditionCol timeCol
## assays(1): expression
## rownames: NULL
## rowData names(1): prot_id
## colnames: NULL
## colData names(3): sample condition timepeptExp[1, 1:2]## class: SilacPeptideExperiment
## dim: 1 2
## metadata(3): conditionCol timeCol proteinCol
## assays(1): expression
## rownames: NULL
## rowData names(2): pept_id prot_id
## colnames: NULL
## colData names(3): sample condition time## subsetting by rows based on rowData
subset(protExp, prot_id == 'A')## class: SilacProteinExperiment
## dim: 1 3
## metadata(2): conditionCol timeCol
## assays(1): expression
## rownames: NULL
## rowData names(1): prot_id
## colnames: NULL
## colData names(3): sample condition timesubset(peptExp, pept_id %in% c('a', 'b'))## class: SilacPeptideExperiment
## dim: 2 3
## metadata(3): conditionCol timeCol proteinCol
## assays(1): expression
## rownames: NULL
## rowData names(2): pept_id prot_id
## colnames: NULL
## colData names(3): sample condition time## quick acces to colData
protExp$sample## [1] "A1" "A2" "A3"peptExp$condition## [1] "A" "A" "A"## combining by columns
cbind(protExp[, 1], protExp[, 2:3])## class: SilacProteinExperiment
## dim: 3 3
## metadata(2): conditionCol timeCol
## assays(1): expression
## rownames: NULL
## rowData names(1): prot_id
## colnames: NULL
## colData names(3): sample condition time## combining by rows
rbind(peptExp[1:3,], peptExp[4:5, ])## class: SilacPeptideExperiment
## dim: 5 3
## metadata(3): conditionCol timeCol proteinCol
## assays(1): expression
## rownames: NULL
## rowData names(2): pept_id prot_id
## colnames: NULL
## colData names(3): sample condition time## combine rows and columns
merge(peptExp[1:3, 1], peptExp[3:5, 2:3])## class: SilacPeptideExperiment
## dim: 5 3
## metadata(3): conditionCol timeCol proteinCol
## assays(1): expression
## rownames: NULL
## rowData names(2): pept_id prot_id
## colnames: NULL
## colData names(3): sample condition time3.2 SilacProteomicsExperiment
The SilacProteomicsExperiment object contains both a SilacProteinExperiment and a SilacPeptideExperiment object. Therefore it can be used to store both levels of information in a single object. The protein and peptide level are aligned/linked by the colData slot and by the linkerDf slot. The linkerDf slot contains a data.frame that indicates which peptides are assigned to each protein and vice versa.
Figure 3: SilacProteomicsExperiment object structure
3.2.1 Object construction
To construct a SilacProteomicsExperiment we need both a SilacProteinExperiment object and a SilacPeptideExperiment object.
ProteomicsExp <- SilacProteomicsExperiment(SilacProteinExperiment = protExp,
SilacPeptideExperiment = peptExp)
ProteomicsExp## class: SilacProteomicsExperiment
## metadata(7): idColProt idColPept ... timeCol proteinCol
## protein:
## |-- dim: 3 3
## |-- assays(1): expression
## |-- rownames: NULL
## |-- rowData names(1): prot_id
## peptide:
## |-- dim: 5 3
## |-- assays(1): expression
## |-- rownames: NULL
## |-- rowData names(2): pept_id prot_id
## colnames: NULL
## colData names(3): sample condition timeIn this case, the levels are only aligned by columns. There is no information on which are the relationships between proteins and peptides. This information can be added in the linkerDf slot. Which contains a data.frame that indicates the relationships between the two levels features. One can be constructed with the buildLinkerDf() function.
## list with the relationships
protein_to_peptide <- list(A = c('a', 'b'), B = c('c'), C = c('d', 'e'))
## function to build the data.frame
linkerDf <- buildLinkerDf(protIDs = LETTERS[1:3],
pepIDs = letters[1:5],
protToPep = protein_to_peptide)
linkerDf## protID pepID protRow pepRow
## 1 A a 1 1
## 2 A b 1 2
## 3 B c 2 3
## 4 C d 3 4
## 5 C e 3 5ProteomicsExp <- SilacProteomicsExperiment(SilacProteinExperiment = protExp,
SilacPeptideExperiment = peptExp,
linkerDf = linkerDf)Linked SilacProteomicsExperiment objects can be useful to apply linked subsetting operations or to recalculate protein level data from peptide data.
3.2.2 Accessor functions
If the slot is general to the SilacProteomicsExperiment object then the same functions as in a SummarizedExperiment can be used.
## colData
colData(ProteomicsExp)## DataFrame with 3 rows and 3 columns
## sample condition time
## <character> <character> <numeric>
## 1 A1 A 1
## 2 A2 A 2
## 3 A3 A 3## linkerDf
linkerDf(ProteomicsExp)## protID pepID protRow pepRow
## 1 A a 1 1
## 2 A b 1 2
## 3 B c 2 3
## 4 C d 3 4
## 5 C e 3 5## metaoptions
metaoptions(ProteomicsExp)## $conditionCol
## [1] "condition"
##
## $timeCol
## [1] "time"
##
## $idColProt
## [1] NA
##
## $idColPept
## [1] NA
##
## $linkedSubset
## [1] TRUE
##
## $subsetMode
## [1] "protein"
##
## $proteinCol
## [1] NABut for names shared between protein and peptide slots, like assays and rowData, there are two options to access them. Using the SummarizedExperiment generic will return a named list with the two elements.
## assays
assays(ProteomicsExp)## $protein
## List of length 1
## names(1): expression
##
## $peptide
## List of length 1
## names(1): expression## rowData
rowData(ProteomicsExp)## $protein
## DataFrame with 3 rows and 1 column
## prot_id
## <character>
## 1 A
## 2 B
## 3 C
##
## $peptide
## DataFrame with 5 rows and 2 columns
## pept_id prot_id
## <character> <character>
## 1 a A
## 2 b A
## 3 c B
## 4 d C
## 5 e COne can use the same functions and just add Prot or Pept as suffixes to specifically access a slot at the protein or the peptide level.
## assays of protein level
assaysProt(ProteomicsExp)## List of length 1
## names(1): expression## assays of peptide level
assaysPept(ProteomicsExp)## List of length 1
## names(1): expression## rowData of protein level
rowDataProt(ProteomicsExp)## DataFrame with 3 rows and 1 column
## prot_id
## <character>
## 1 A
## 2 B
## 3 C## rowData of peptide level
rowDataPept(ProteomicsExp)## DataFrame with 5 rows and 2 columns
## pept_id prot_id
## <character> <character>
## 1 a A
## 2 b A
## 3 c B
## 4 d C
## 5 e CFinally, to extract the SilacProteinExperiment and SilacPeptideExperiment one can use the ProtExp and PeptExp functions.
ProtExp(ProteomicsExp)## class: SilacProteinExperiment
## dim: 3 3
## metadata(2): conditionCol timeCol
## assays(1): expression
## rownames: NULL
## rowData names(1): prot_id
## colnames: NULL
## colData names(3): sample condition timePeptExp(ProteomicsExp)## class: SilacPeptideExperiment
## dim: 5 3
## metadata(3): conditionCol timeCol proteinCol
## assays(1): expression
## rownames: NULL
## rowData names(2): pept_id prot_id
## colnames: NULL
## colData names(3): sample condition time3.2.3 Subsetting and aggregation
When subsetting row-wise on a SilacProteomicsExperiment there will be two main questions:
- To which level (protein or peptide) is the subset applied?
- Is the subset from one level applied also to the other? For example, when I subset a protein, do I also want to subset also only those peptides related to that protein.
The answer to these questions is written in the metaoptions values. The first one is subsetMode which can be protein or peptide. The second one is linkedSubset, which can be TRUE or FALSE. Moreover, if the latter is set to TRUE then the idColProt and idColPept will also be required.
Here are some examples of using the [ in different situations.
Subset at protein level without affecting the peptide level.
## indicate which rowDat columns have unique ids for proteins and peptides
metaoptions(ProteomicsExp)[['idColProt']] <- 'prot_id'
metaoptions(ProteomicsExp)[['idColPept']] <- 'pept_id'
## indicate that we want to apply the subset at protein level
metaoptions(ProteomicsExp)[['subsetMode']] <- 'protein'
## and not extend it to the peptide level
metaoptions(ProteomicsExp)[['linkedSubset']] <- FALSE
ProteomicsExp[1:2,]## class: SilacProteomicsExperiment
## metadata(7): conditionCol timeCol ... subsetMode proteinCol
## protein:
## |-- dim: 2 3
## |-- assays(1): expression
## |-- rownames: NULL
## |-- rowData names(1): prot_id
## peptide:
## |-- dim: 5 3
## |-- assays(1): expression
## |-- rownames: NULL
## |-- rowData names(2): pept_id prot_id
## colnames: NULL
## colData names(3): sample condition timeNote that the protein level has two proteins while the peptide level kept all five peptides.
Subset at protein level and extending the subset to the peptide level.
## to extend we set the metaoption to TRUE
metaoptions(ProteomicsExp)[['linkedSubset']] <- TRUE
ProteomicsExp[1:2,]## class: SilacProteomicsExperiment
## metadata(7): conditionCol timeCol ... subsetMode proteinCol
## protein:
## |-- dim: 2 3
## |-- assays(1): expression
## |-- rownames: NULL
## |-- rowData names(1): prot_id
## peptide:
## |-- dim: 3 3
## |-- assays(1): expression
## |-- rownames: NULL
## |-- rowData names(2): pept_id prot_id
## colnames: NULL
## colData names(3): sample condition timeIn this case the protein level has two proteins and the peptide level has also been subset to the three peptides that are assigned to the two proteins in the linkerDf.
The same process can also be applied at peptide level.
## indicate that we want to apply the subset at protein level
metaoptions(ProteomicsExp)[['subsetMode']] <- 'peptide'
## to extend we set the metaoption to TRUE
metaoptions(ProteomicsExp)[['linkedSubset']] <- TRUE
ProteomicsExp[1:2,]## class: SilacProteomicsExperiment
## metadata(7): conditionCol timeCol ... subsetMode proteinCol
## protein:
## |-- dim: 1 3
## |-- assays(1): expression
## |-- rownames: NULL
## |-- rowData names(1): prot_id
## peptide:
## |-- dim: 2 3
## |-- assays(1): expression
## |-- rownames: NULL
## |-- rowData names(2): pept_id prot_id
## colnames: NULL
## colData names(3): sample condition timeIn this case, the first two peptides, which belong to the first protein, are selected along with that protein.
Other subset operations can be done using subsetProt() and subsetPep(), which call the generic subset() on the rowData slot of the protein and peptide level respectively. Note that subsetProt() and subsetPep() will behave differently depending on the metaoption linkedSubset as seen above with [.
## without linked Subset
metaoptions(ProteomicsExp)[['linkedSubset']] <- FALSE
subsetProt(ProteomicsExp, prot_id == 'B')## class: SilacProteomicsExperiment
## metadata(7): conditionCol timeCol ... subsetMode proteinCol
## protein:
## |-- dim: 1 3
## |-- assays(1): expression
## |-- rownames: NULL
## |-- rowData names(1): prot_id
## peptide:
## |-- dim: 5 3
## |-- assays(1): expression
## |-- rownames: NULL
## |-- rowData names(2): pept_id prot_id
## colnames: NULL
## colData names(3): sample condition time## with linked Subset
metaoptions(ProteomicsExp)[['linkedSubset']] <- TRUE
subsetProt(ProteomicsExp, prot_id == 'B')## class: SilacProteomicsExperiment
## metadata(7): conditionCol timeCol ... subsetMode proteinCol
## protein:
## |-- dim: 1 3
## |-- assays(1): expression
## |-- rownames: NULL
## |-- rowData names(1): prot_id
## peptide:
## |-- dim: 1 3
## |-- assays(1): expression
## |-- rownames: NULL
## |-- rowData names(2): pept_id prot_id
## colnames: NULL
## colData names(3): sample condition timeFinally, cbind, rbind and merge can be used for aggregation.
For cbind the number of proteins and peptides must be the same and they are expected to be in the same order.
## cbind
cbind(ProteomicsExp[, 1], ProteomicsExp[, 2])## class: SilacProteomicsExperiment
## metadata(7): conditionCol timeCol ... subsetMode proteinCol
## protein:
## |-- dim: 3 2
## |-- assays(1): expression
## |-- rownames: NULL
## |-- rowData names(1): prot_id
## peptide:
## |-- dim: 5 2
## |-- assays(1): expression
## |-- rownames: NULL
## |-- rowData names(2): pept_id prot_id
## colnames: NULL
## colData names(3): sample condition timeFor rbind the number of samples must be the same and they are expected to be in the same order.
## rbind
rbind(ProteomicsExp[1:2,], ProteomicsExp[3,])## class: SilacProteomicsExperiment
## metadata(7): conditionCol timeCol ... subsetMode proteinCol
## protein:
## |-- dim: 2 3
## |-- assays(1): expression
## |-- rownames: NULL
## |-- rowData names(1): prot_id
## peptide:
## |-- dim: 3 3
## |-- assays(1): expression
## |-- rownames: NULL
## |-- rowData names(2): pept_id prot_id
## colnames: NULL
## colData names(3): sample condition timemerge can be used when samples have to be aggregated in one object but the proteins/peptides are not the same. It requires IDs that can be matched between experiments.
## merge
merge(ProteomicsExp[1:3, 1], ProteomicsExp[3:4, 2:3])## class: SilacProteomicsExperiment
## metadata(7): conditionCol timeCol ... subsetMode proteinCol
## protein:
## |-- dim: 3 3
## |-- assays(1): expression
## |-- rownames: NULL
## |-- rowData names(1): prot_id
## peptide:
## |-- dim: 4 3
## |-- assays(1): expression
## |-- rownames: NULL
## |-- rowData names(2): pept_id prot_id
## colnames: NULL
## colData names(3): sample condition time3.3 metaoptions
The metaoptions are a set of “option” values used to indicate where critical analysis information is required. The idea is that these values are defined at the beginning of the analysis so that it is not required to give them as arguments in each function call. Nevertheless, all the analysis functions can have these values passed as arguments. There are several metaoptions values by default, some of them are shared between the three classes while others are unique.
The following are related to data analysis:
conditionCol: which column incolDataindicates the different experiment conditions.timeCol: which column incolDataindicates the different timepoints of the experiment.proteinCol: which column inrowDataindicates the assigned protein to a peptide. (unique toSilacPeptideExperiment)
The following are related to SilacProteomicsExperiment subsetting operations:
idColProt: which column inrowDataProtindicates unique protein IDs. (unique toSilacProteomicsExperiment)idColPept: which column inrowDataPeptindicates unique peptide IDs. (unique toSilacProteomicsExperiment)linkedSubset: logical that indicates if the subsetting operation should be applied to both data levels (protein and peptide). (unique toSilacProteomicsExperiment)subsetMode: which level should be used for reference when subsetting (protein or peptide). (unique toSilacProteomicsExperiment)
These can be extended by the user to accommodate other experiment design and data analysis operations.
4 Example data
To demonstrate the use of the package functions, a reduced version of the pulsed-SILAC dataset from Visscher et al. (2016) has been built into a SilacProteomicsExperiment object. It is stored in an object named wormsPE. This object has been constructed from the output files from MaxQuant ‘proteinGroups.txt’ and ‘peptides.txt’.
wormsPE## class: SilacProteomicsExperiment
## metadata(7): idColProt idColPept ... timeCol proteinCol
## protein:
## |-- dim: 250 14
## |-- assays(4): int_total int_light int_heavy ratio
## |-- rownames: NULL
## |-- rowData names(22): Protein.IDs Majority.protein.IDs ...
## Peptide.is.razor protein_id
## peptide:
## |-- dim: 3574 14
## |-- assays(4): int_total int_light int_heavy ratio
## |-- rownames: NULL
## |-- rowData names(46): Sequence N.term.cleavage.window ... id
## Protein.group.IDs
## colnames: NULL
## colData names(2): line timepointThe dataset contains only the first 250 proteins and their corresponding peptides. It has 4 assays with the total intensity, light isotope and heavy isotope intensities and heavy/light isotope ratio. In the experiment there are 2 worm cell lines: OW40, which express \(\alpha\)-synuclein-YFP (model for Parkinson’s Disease) and OW450, which express YFP only. Worms were harvested after 4h, 6h, 8h, 13h, 24h, 28h and 32h of light to heavy isotope medium transition. Note that the analysis of the data presented in the paper by Visscher et al was not performed using this package. However, all data in that paper has been reanalyzed using this package, which resulted in general the same but also more extended conclusions.
5 Filtering
5.1 Measurements across time
To reliably model a protein’s turnover, several timepoints are required. Ideally a protein would be measured in all the timepoints of the experiment, but this is rarely the case. To count how many proteins/peptides have been measured in each sample the barplotCounts() can be used.
barplotCounts(wormsPE, assayName = 'ratio')
There is some variability between samples but nothing too extreme. More importantly is knowing whether a protein has been measured across different timepoints. The function barplotTimeCoverage() can be used to visualize in how many timepoints each protein has been measured per condition.
barplotTimeCoverage(wormsPE, assayName = 'ratio')
As it can be seen, most of the proteins are not measured in all timepoints. In this case there is a majority for proteins that have not been measured in any timepoint, this is because the dataset has been truncated for only the OW40 and OW450 lines. The original search was one with additional worm lines and the missing proteins were detected in the lines excluded from this vignette.
To filter proteins that have not been detected in sufficient timepoints the filterByMissingTimepoints() function can be used. With maxMissing the amount of missing values can be tuned. With strict proteins have to meet the maxMissing criteria only in one condition or in all.
wormsPE2 <- filterByMissingTimepoints(wormsPE,
assayName = 'ratio',
maxMissing = 2,
strict = FALSE)
barplotTimeCoverage(wormsPE2, assayName = 'ratio')
Because strict is set to FALSE, there are still some proteins that have been detected in less than 5 timepoints in one of the conditions. To visualize the overlap between conditions, the upsetTimeCoverage() function can be used. This can be especially useful when more the experiment contains more than two conditions. Here the overlap of proteins is shown.
upsetTimeCoverage(ProtExp(wormsPE2),
assayName = 'ratio',
maxMissing = 2)
In this case there are 68 proteins that have been measured in at least 5 out of 7 timepoints in both conditions. These would be proteins for which turnover models can be compared between conditions.
Filtering by missing timepoints can also be done at peptide level if desired by changing subsetMode to peptide.
5.2 Other filters
Other filters can be easily applied using the subset method. For example, proteins with a low number of unique peptides can be filtered out.
subsetProt(wormsPE, Unique.peptides > 2)Also, proteins that are known as common contaminants or identified using the reverse database can be filtered out.
subsetProt(wormsPE, Potential.contaminant != '+')
subsetProt(wormsPE, Reverse != '+')6 Ratio and fraction calculation
To model protein turnover the isotope fractions from each timepoint are used. The isotope fraction can be calculated in many ways. A simple approach is to derive them from isotope ratios, which many raw mass spectrometry data analysis software report. If not, one can use the calculateIsotopeRatio() function, which will add a new assay named ratio to the object. In this example, the ratio of newly synthesized protein (heavy isotope) over old protein (light isotope) will be used.
## calculate the ratio of new istope over old isotope
wormsPE <- calculateIsotopeRatio(x = wormsPE,
newIsotopeAssay = 'int_heavy',
oldIsotopeAssay = 'int_light')\[ratio = \frac{isotope_{A}}{isotope_{B}}\]
And then the fraction can be calculated as follows:
\[fraction_{A} = \frac{ratio}{1 + ratio}\]
This can be easily done using the calculateIsotopeFraction() function, which adds a new assay named fraction. In this case the heavy isotope fraction is calculated, the light isotope fraction would just be 1-fractionA.
wormsPE <- calculateIsotopeFraction(wormsPE, ratioAssay = 'ratio')
assaysProt(wormsPE)## List of length 5
## names(5): int_total int_light int_heavy ratio fractionassaysPept(wormsPE)## List of length 5
## names(5): int_total int_light int_heavy ratio fractionUnfortunately, this approach might might lead to the loss some values because mass spectrometry has a bias towards the measurement of high abundance peptides. For example: if a protein has a very slow turnover rate, it might be that at the earliest timepoint, the new isotope signal was below the detection limit and a ratio could not be calculated. To also account for these proteins the calculateIsotopeFraction function has additional arguments to indicate which assays contain each isotope’s expression data and which timepoints are considered “early” or “late”.
wormsPE <- calculateIsotopeFraction(wormsPE,
newIsoAssay = 'int_heavy',
oldIsoAssay = 'int_light',
earlyTimepoints = 1,
lateTimepoints = 7)7 Plotting assays
To visualize data stored in the assays slot there are two functions plotDistributionAssay() and scatterCompareAssays(). The first one will plot the distributions of the data for a selected assay for each timepoint and condition. The latter will plot each protein/peptide value of a condition against the other for each timepoint.
7.1 plotDistributionAssay()
plotDistributionAssay(wormsPE, assayName = 'fraction')
From this plot it can already be seen that the OW450 line has a much faster heavy label incorporation than the OW40 line.
7.2 scatterCompareAssays()
For scatterCompareAssays() it is necessary to indicate which conditions should be compared. To make it simpler, only the protein data will be used.
protPE <- ProtExp(wormsPE)
scatterCompareAssays(x = protPE,
conditions = c('OW40', 'OW450'),
assayName = 'ratio')
This plot shows that the OW450 has a faster incorporation of heavy label than the OW40 line.
scatterCompareAssays(x = protPE,
conditions = c('OW40', 'OW450'),
assayName = 'int_total')
This plot shows that both the OW450 and OW40 have similar total protein expression over time.
8 Model protein turnover
To estimate protein turnover rate the modelTurnover() function can be used. Parameter fitting is done using the nls function, or the nlrob function from robustbase for robust modelling. The formula, start and ... arguments are passed into these functions. By default, an exponential decay model is used, but it has been shown that some proteins follow other turnover kinetics McShane et al. (2016).
modelList <- modelTurnover(x = wormsPE,
assayName = 'fraction',
formula = 'fraction ~ 1 - exp(-k*t)',
start = list(k = 0.02),
mode = 'protein',
returnModel = TRUE)8.1 Output of modelTurnover()
The output of modelTurnover() is a list with the main model metrics:
residuals: a matrix of the same dimensions asxwith the model residuals.stderror: a matrix with the standard error of each model.param_values: a matrix with the estimated parameter values of each model.param_pval: a matrix with the p-value of each parameter of each model.param_tval: a matrix with the t-statistic of each parameter of each model.param_stderror: a matrix with the standard error of each parameter of each model.
Models that fail to converge have NA as their output metrics.
There are more options to tune modelTurnover(), such as: robust, to use robust modelling; returnModel, which will return the actual model objects besides the metrics; verbose, to output a progress bar. For more information please refer to the manual.
8.2 Calculating protein half-life
To calculate protein half-life one has to separate the time variable on to one side of the equation and solve it. For this particular exponential decay model that has been applied it is quite simple. The new equation would be:
\[t = \frac{log(0.5)}{-k}\]
The half-life, of each protein can be calculated using this formula. Here it is calculated and added to the modelList object. In this way, the following plots can also be used on the protein half-life data.
modelList[['half_life']] <- log(0.5)/(-modelList$param_values$k)8.3 Plots
The output of modelTurnover() with returnModel = TRUE can be used as input in the following functions to plot either individual protein/peptides models or the overall experiment distributions.
All the following function return objects of class ggplot, therefore their theme, scale, labels, etc. can be customized further customized. Moreover, if the argument returnDataFrame is set to TRUE, the data.frame used in the plot function will be returned instead.
8.3.1 Individual models
To plot individual models the plotIndividualModel() function can be used. As input it requires the Experiment object that was used in the modelTurnover() call and its output. This function also requires returnModel = TRUE.
modelList <- modelTurnover(x = wormsPE,
assayName = 'fraction',
formula = 'fraction ~ 1 - exp(-k*t)',
start = list(k = 0.02),
mode = 'protein',
returnModel = TRUE)
plotIndividualModel(x = wormsPE,
modelList = modelList,
num = 2)
The plot shows the quantification data as dots and the fitted model as a curve. If the models are based on grouped peptide data then the values of each peptide are plotted as dots.
## to indicate which column of rowDataPept indicates the assigned protein
metaoptions(wormsPE)[['proteinCol']] <- 'Protein.group.IDs'
modelList <- modelTurnover(x = wormsPE,
assayName = 'fraction',
formula = 'fraction ~ 1 - exp(-k*t)',
start = list(k = 0.02),
mode = 'grouped',
returnModel = TRUE)## Modelling peptides grouped by proteinplotIndividualModel(x = wormsPE,
modelList = modelList,
num = 2)
8.3.2 Distributions
To plot the overall distributions of the model metrics per condition the plotDistributionModel function can be used. This will show global changes between conditions and/or can be used for quality control. Some metrics that might be interesting to check are the R2 error of the models, the estimated parameters, the residuals and the weights (only for robust modelling).
In this case, both the outputs of modelTurnover() with returnModel = TRUE or with returnModel = FALSE will work.
8.3.2.1 R2 error
modelList <- modelTurnover(x = wormsPE,
assayName = 'fraction',
formula = 'fraction ~ 1 - exp(-k*t)',
start = list(k = 0.02),
mode = 'protein',
robust = TRUE,
returnModel = TRUE)This plot shows the distribution of the error of all the models for each condition.
plotDistributionModel(modelList = modelList,
value = 'stderror',
plotType = 'density')
8.3.2.2 Turnover rate
This plot shows the distribution of the values for every fitted parameter. In this case there is only one: the turnover rate (k).
plotDistributionModel(modelList = modelList,
value = 'param_values',
plotType = 'density')
8.3.2.3 Residuals
This plot shows the distribution of residuals at each timepoint for each condition.
plotDistributionModel(modelList = modelList,
value = 'residuals',
plotType = 'density')
8.3.2.4 Weights
This plot shows the distribution of weights, from robust modelling, at each timepoint for each condition. This is only available if robust modelling is used.
plotDistributionModel(modelList = modelList,
value = 'weights',
plotType = 'density')
8.3.2.5 Half-lives
This plot shows the distribution of half-lives for each condition.
plotDistributionModel(modelList = modelList,
value = 'half_life',
plotType = 'density')
8.3.3 Comparing conditions
To plot protein/peptide values and compare between conditions the scatterCompareModels() function can be used. It works like the scatterCompareAssays() function. The two conditions to be compared must be indicated as well as the desired metric.
scatterCompareModels(modelList = modelList,
conditions = c('OW40', 'OW450'),
value = 'param_values')
scatterCompareModels(modelList = modelList,
conditions = c('OW40', 'OW450'),
value = 'stderror')
8.4 Comparing models
It might be that some proteins follow a specific turnover model, while other follow a different one. In that case, one can run modelTurnover() for each different model and then compare the models for each protein. One metric often used to compare models is the Akaike Information Criteria (AIC). It can be calculated using the calculateAIC() function. Afterwards the probability of each model relative to the rest can be calculated with the compareAIC() function.
8.4.1 Calculating the AIC
To calculate the AIC the output from modelTurnover() can be used. The formula used to calculate it is the following:
\[AIC = 2k - 2ln(logLik)\]
There is also a formula that applies a small sample size correction:
\[AICc = AIC + \frac{2k(k + 1)}{n - k - 1}\]
Where k is the number of parameters and n is the number of observations (timepoints in this case).
modelList <- calculateAIC(modelList, smallSampleSize = TRUE)The output will be the same given list plus an additional matrix with the AIC values for each model and condition.
names(modelList)## [1] "residuals" "stderror" "param_values" "param_pval"
## [5] "param_tval" "param_stderror" "weights" "models"
## [9] "half_life" "AIC"plotDistributionModel(modelList = modelList, value = 'AIC')
8.4.2 Relative model probabilities
After the AICs have been calculated, the probability of one model relative to the others can be calculated. This is done using the compareAIC function. This probability can be calculated using the following formula:
\[\prod AIC_{i} = \frac{exp(\frac{AIC_{min}-AIC_{i}}{2})}{\sum_{j}exp(\frac{AIC_{min}-AIC_{j}}{2})} \]
The compareAIC function uses as input an indefinite number of modelLists that have been processed through the calculateAIC function, and it returns a list of matrices with the relative probability of each model (columns) for each protein/peptide (rows).
modelList1 <- modelTurnover(x = wormsPE,
assayName = 'fraction',
formula = 'fraction ~ 1 - exp(-k*t)',
start = list(k = 0.02),
mode = 'protein',
robust = FALSE,
returnModel = TRUE)
modelList1 <- calculateAIC(modelList = modelList1,
smallSampleSize = TRUE)
modelList2 <- modelTurnover(x = wormsPE,
assayName = 'fraction',
formula = 'fraction ~ 1 - exp(-k*t) + b',
start = list(k = 0.02, b = 0),
mode = 'protein',
robust = FALSE,
returnModel = TRUE)
modelList2 <- calculateAIC(modelList = modelList2,
smallSampleSize = TRUE)Here is a small example in which two different models have been applied.
modelProbabilities <- compareAIC(modelList1, modelList2)
plotDistributionModel(modelList = modelProbabilities,
value = 'aicprobabilities',
returnDataFrame = FALSE)
The plot shows that model1 is the preferred model of the two in both conditions for most proteins, but there are some that have a better model with the b parameter.
9 Cell growth estimation
During most pulsed-SILAC experiment cells will keep dividing. This means that protein synthesis will be greater than protein degradation because biomass increases. When comparing conditions in which cells divide at similar rates this effect can be neglected. But if cell growth is different between conditions then steady-state protein turnover will be obfuscated by the difference in protein synthesis due to cell growth.
This issue can be corrected by using a turnover model which takes cell growth into consideration. For example, by adding the cell growth rate into the model (where tcc is the cell division average time):
\[f(H) = 1 - e^{-(k + \frac{log 2}{t_{cc}})t}\]
If this information is not available, the estimateCellGrowth() function can be used. This function will fit an exponential degradation model based on the n most stable proteins (proteins which are measured in all time points and have the slowest new isotope incorporation). The rationale behind this approach is that the most stable proteins are very slowly degraded (for example structural proteins), but new proteins need to be synthesized for the daughter cells. Therefore, these proteins can be used to infer cell growth.
9.1 Example data
For the current dataset, cell division was not an issue since cells in the adult C. elegans soma no longer divide. To illustrate the effects of differential cell division on the output, data from mouse embryonic fibroblasts (MEFs) cultured with and without serum will be used. MEFs cultured with serum will divide at a “normal” rate while MEFs cultured without serum will hardly divide and hence the total biomass will be quite different between samples at the end of the experiment.
mefPE## class: SilacProteinExperiment
## dim: 5223 10
## metadata(2): conditionCol timeCol
## assays(2): ratio fraction
## rownames: NULL
## rowData names(3): Protein.IDs Gene.names Fasta.headers
## colnames: NULL
## colData names(2): line timepointThis can be appreciated if the heavy label fraction is plotted over time for the two conditions.
plotDistributionAssay(mefPE, assayName = 'fraction')
9.2 Most stable proteins
To extract the most stable proteins the mostStable() function can be used. This function will rank the incorporation of new label across time and conditions, and then select the top n proteins/peptides.
stablePE <- mostStable(mefPE, assayName = 'fraction', n = 50)
stablePE## class: SilacProteinExperiment
## dim: 50 10
## metadata(2): conditionCol timeCol
## assays(2): ratio fraction
## rownames: NULL
## rowData names(3): Protein.IDs Gene.names Fasta.headers
## colnames: NULL
## colData names(2): line timepoint9.3 Estimating cell growth
The stable protein set can then be used to estimate the growth rate, assuming that the incorporation of heavy
isotope in these proteins is predominantly due to increased biomass rather than replacement. The modelTurnover function can be used again since it follows the same principles. In the following example, the same exponential degradation model is applied, but instead of the turnover rate the doubling time parameter is used.
stableModels <- modelTurnover(x = stablePE,
assayName = 'fraction',
formula = 'fraction ~ 1 - exp(-(log(2)/tc)*t)',
start = list(tc = 20),
mode = 'protein',
robust = FALSE,
returnModel = FALSE)
plotDistributionModel(stableModels, value = 'param_values', plotType = 'boxplot')
Because the doubling time will be modeled from 50 different proteins, the doubling time needs to be summarized. This can be done by taking for example the mean form these 50 models.
apply(stableModels$param_values$tc, 2, mean)## NoSerum Serum
## 93.15081 25.10730Note that there is a clear difference in the doubling times comparing the two conditions.
9.4 Comparing turnover rates
Next the turnover rates can be calculated using the doubling time correction model. Because each condition has a different doubling time, the models have to be run separately.
modelsNoSerum <- modelTurnover(x = mefPE[, 1:5],
assayName = 'fraction',
formula = 'fraction ~ 1 - exp(-(0.0074 + k)*t)',
start = list(k = 0.02),
mode = 'protein',
robust = FALSE,
returnModel = TRUE)
modelsSerum <- modelTurnover(x = mefPE[, 6:10],
assayName = 'fraction',
formula = 'fraction ~ 1 - exp(-(0.0276 + k)*t)',
start = list(k = 0.02),
mode = 'protein',
robust = FALSE,
returnModel = TRUE)
modelsMef <- mergeModelsLists(modelsNoSerum, modelsSerum)To compare the output from the models without correction for increased biomass, the turnover is modeled without the doubling time correction.
modelsNoCorrect <- modelTurnover(x = mefPE,
assayName = 'fraction',
formula = 'fraction ~ 1 - exp(-(k)*t)',
start = list(k = 0.02),
mode = 'protein',
robust = FALSE,
returnModel = TRUE)Finally, the distributions of the turnover rates can be plotted for each case.
With doubling time correction:
plotDistributionModel(modelList = modelsMef,
value = 'param_values',
plotType = 'density') ## Picking joint bandwidth of 0.00279
Without doubling time correction:
plotDistributionModel(modelList = modelsNoCorrect,
value = 'param_values',
plotType = 'density') ## Picking joint bandwidth of 0.00279
10 Isotope recycling estimation
Once a protein is degraded, its amino acids can be recycled to synthesize new proteins. This process will lead to an underestimation of protein turnover. For example, light label amino acids will be used to synthesize new proteins, but will be interpreted as being old proteins. This results in an overall overestimation of protein half-life.
To estimate how much “old” label is still being used in new proteins, miss-cleaved peptides can be used. If a miss-cleaved peptide contains both light and heavy label, it means that old label was used for a new protein. The amount of light label recycling can be estimated using the following formula:
\[ \frac{1}{2\frac{Intensity_{NewNew}}{Intensity_{NewOld}} + 1} = Old (Fraction)\]
Unfortunately MaxQuant does not search for mixed isotope peptides by default. Therefore a second search has to be done if one wants to obtain this information. But this could be different for other platforms.
10.1 Adding the information
To add the information on muss-cleaved peptides the addMiscleavedPeptides() function can be used. This function adds the data from the second search into a SilacPeptideExperiment or a SilacProteomicsExperiment. If the analysis has been done using protein data, then a SilacPeptideExperiment object with this information is created. In this example the SilacProteinExperiment object will be used because the peptide data in wormsPE is not complete.
protPE <- ProtExp(wormsPE)
missPE <- addMisscleavedPeptides(x = protPE,
newdata = recycleLightLysine,
idColPept = 'Sequence',
modCol = 'Modifications',
dataCols = c(18:31))
assays(missPE)## List of length 2
## names(2): 2 Copy of Lys8 Copy of Lys8There are two assays in the missPE object. These contain the intensities for peptides that contain two heavy isotope lysines (new label) and intensities for peptides that contain one heavy and one light isotope lysines.
10.2 Calculating light label recycling
To calculate the amount of light label used in protein synthesis the calculateOldIsotopePool() function can be used. This function applies the formula mentioned above and adds a new assay with the name of oldIsotopePool.
names(assays(missPE))[1:2] <- c('int_lys8lys8', 'int_lys8lys0')
missPE <- calculateOldIsotopePool(x = missPE, 'int_lys8lys8', 'int_lys8lys0')Finally the distributions of these pools can be shown in a boxplot.
plotDistributionAssay(missPE, assayName = 'oldIsotopePool')## Warning: Removed 8722 rows containing non-finite values (stat_boxplot).
The supplemental figure 6B of Visscher et al. (2016) can be reproduced using the above method.
11 Interoperability with other packages
The main objective of this package is proteomics turnover analysis, but there are other types of analysis that can be done with the same data: enrichment analysis, differential protein expression, etc. To facilitate the interoperability from SilacProteinExperiment and SilacPeptideExperiment objects there are coercion methods available to the more general classes such as SummarizedExperiment and data.frame.
For example, the DEP package uses the SummarizedExperiment class for their differential protein expression analysis functions.
protExp <- ProtExp(wormsPE)
as(protExp, 'SummarizedExperiment')## class: SummarizedExperiment
## dim: 250 14
## metadata(2): conditionCol timeCol
## assays(5): int_total int_light int_heavy ratio fraction
## rownames: NULL
## rowData names(22): Protein.IDs Majority.protein.IDs ...
## Peptide.is.razor protein_id
## colnames: NULL
## colData names(2): line timepoint12 Session info
sessionInfo()## R version 4.0.0 (2020-04-24)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 18.04.4 LTS
##
## Matrix products: default
## BLAS: /home/biocbuild/bbs-3.11-bioc/R/lib/libRblas.so
## LAPACK: /home/biocbuild/bbs-3.11-bioc/R/lib/libRlapack.so
##
## locale:
## [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
## [3] LC_TIME=en_US.UTF-8 LC_COLLATE=C
## [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
## [7] LC_PAPER=en_US.UTF-8 LC_NAME=C
## [9] LC_ADDRESS=C LC_TELEPHONE=C
## [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
##
## attached base packages:
## [1] stats graphics grDevices utils datasets methods base
##
## other attached packages:
## [1] pulsedSilac_1.2.0 BiocStyle_2.16.0
##
## loaded via a namespace (and not attached):
## [1] Rcpp_1.0.4.6 lattice_0.20-41
## [3] assertthat_0.2.1 digest_0.6.25
## [5] R6_2.4.1 MuMIn_1.43.17
## [7] GenomeInfoDb_1.24.0 plyr_1.8.6
## [9] ggridges_0.5.2 stats4_4.0.0
## [11] evaluate_0.14 ggplot2_3.3.0
## [13] pillar_1.4.3 zlibbioc_1.34.0
## [15] rlang_0.4.5 magick_2.3
## [17] S4Vectors_0.26.0 R.utils_2.9.2
## [19] R.oo_1.23.0 Matrix_1.2-18
## [21] rmarkdown_2.1 labeling_0.3
## [23] stringr_1.4.0 RCurl_1.98-1.2
## [25] munsell_0.5.0 DelayedArray_0.14.0
## [27] compiler_4.0.0 xfun_0.13
## [29] pkgconfig_2.0.3 BiocGenerics_0.34.0
## [31] htmltools_0.4.0 tidyselect_1.0.0
## [33] SummarizedExperiment_1.18.0 tibble_3.0.1
## [35] gridExtra_2.3 GenomeInfoDbData_1.2.3
## [37] bookdown_0.18 IRanges_2.22.0
## [39] matrixStats_0.56.0 crayon_1.3.4
## [41] dplyr_0.8.5 bitops_1.0-6
## [43] R.methodsS3_1.8.0 grid_4.0.0
## [45] nlme_3.1-147 gtable_0.3.0
## [47] lifecycle_0.2.0 magrittr_1.5
## [49] scales_1.1.0 stringi_1.4.6
## [51] farver_2.0.3 XVector_0.28.0
## [53] reshape2_1.4.4 robustbase_0.93-6
## [55] ellipsis_0.3.0 vctrs_0.2.4
## [57] cowplot_1.0.0 tools_4.0.0
## [59] taRifx_1.0.6.2 Biobase_2.48.0
## [61] glue_1.4.0 DEoptimR_1.0-8
## [63] purrr_0.3.4 parallel_4.0.0
## [65] yaml_2.2.1 colorspace_1.4-1
## [67] BiocManager_1.30.10 UpSetR_1.4.0
## [69] GenomicRanges_1.40.0 knitr_1.28References
Doherty, Mary K, Colin Whitehead, Heather Mccormack, Simon J Gaskell, and Robert J Beynon. 2005. “Proteome dynamics in complex organisms : Using stable isotopes to monitor individual protein turnover rates,” 522–33. https://doi.org/10.1002/pmic.200400959.
McShane, Erik, Celine Sin, Henrik Zauber, Jonathan N. Wells, Neysan Donnelly, Xi Wang, Jingyi Hou, et al. 2016. “Kinetic Analysis of Protein Stability Reveals Age-Dependent Degradation.” Cell 167 (3). Elsevier Inc.:803–815.e21. https://doi.org/10.1016/j.cell.2016.09.015.
Pratt, Julie M, June Petty, Isabel Riba-garcia, Duncan H L Robertson, Simon J Gaskell, Stephen G Oliver, and Robert J Beynon. 2002. “Dynamics of Protein Turnover , a Missing Dimension in Proteomics *,” 579–91. https://doi.org/10.1074/mcp.M200046-MCP200.
Schwanhäusser, Björn, Manfred Gossen, Gunnar Dittmar, and Matthias Selbach. 2009. “Global analysis of cellular protein translation by pulsed SILAC,” 205–9. https://doi.org/10.1002/pmic.200800275.
Visscher, Marieke, Sasha De Henau, Mattheus H.E. Wildschut, Robert M. van Es, Ineke Dhondt, Helen Michels, Patrick Kemmeren, et al. 2016. “Proteome-wide Changes in Protein Turnover Rates in C. elegans Models of Longevity and Age-Related Disease.” Cell Reports 16 (11). ElsevierCompany.:3041–51. https://doi.org/10.1016/j.celrep.2016.08.025.