This R package provides an implementation of the MaxLFQ algorithm by Cox et al. (2014) in a comprehensive pipeline for DIA-MS (Pham et al. 2020). It also offers options for protein quantification using the N most intense fragment ions, using all fragment ions, and the Tukey's median polish algorithm. In general, the tool can be used to integrate multiple proportional observations into a single quantitative value.
Citation
Pham TV, Henneman AA, Jimenez CR. iq: an R package to estimate relative protein abundances from ion quantification in DIA-MS-based proteomics, Bioinformatics 2020 Apr 15;36(8):2611-2613. https://doi.org/10.1093/bioinformatics/btz961
The package is hosted on CRAN. It is best to install from within R.
install.packages("iq")
See a recent example for processing a Spectronaut output.
Or an older vignette for processing output from Spectronaut, OpenSWATH and MaxQuant with some visualization.
A blog post on converting a .parquet file to a .tsv file.
The package can be loaded in the usual manner
library("iq")
To process a DIA-NN output
For version of DIA-NN prior to 2.0, the following is an iq function call to filter on the Q.Value, PG.Q.Value, Lib.Q.Value, and Lib.PG.Q.Value for a match-between run (MBR) DIA-NN search as discussed here.
process_long_format("report.tsv",
sample_id = "Run",
intensity_col = "Fragment.Quant.Raw",
output_filename = "report-pg-global.txt",
annotation_col = c("Protein.Names", "Genes"),
filter_double_less = c("Q.Value" = "0.01", "PG.Q.Value" = "0.05",
"Lib.Q.Value" = "0.01", "Lib.PG.Q.Value" = "0.01"))
DIA-NN version 2.0 uses the parquet data format for output. We can use the R package arrow to read the data. However, the fragment intensities are not reported by the default setting. To perform quantification using MS/MS fragments, one must switch on the --export-quant option. Then we can use R to create a .tsv as an intermediate step as follows
require("arrow")
# if the package "arrow" is not available, you can install it by
# install.packages("arrow")
raw <- arrow::read_parquet("report.parquet")
# create a new column called "Intensities"
raw$Intensities = paste(raw$Fr.0.Quantity, raw$Fr.1.Quantity, raw$Fr.2.Quantity,
raw$Fr.3.Quantity, raw$Fr.4.Quantity, raw$Fr.5.Quantity,
raw$Fr.6.Quantity, raw$Fr.7.Quantity, raw$Fr.8.Quantity,
raw$Fr.9.Quantity, raw$Fr.10.Quantity, raw$Fr.11.Quantity, sep = ";")
write.table(raw, "report.tsv", sep = "\t", row.names = FALSE, quote = FALSE)
# using the new column "Intensities"
iq::process_long_format("report.tsv",
output_filename = "report-protein-group.txt",
sample_id = "Run",
intensity_col = "Intensities",
annotation_col = c("Protein.Ids","Protein.Names", "Genes"),
filter_double_less = c("Q.Value" = "0.01", "PG.Q.Value" = "0.05",
"Lib.Q.Value" = "0.01",
"Lib.PG.Q.Value" = "0.01"))
Alternatively, we can use the aggregated intensities in Precursor.Normalisedas discussed here.
iq::process_long_format(arrow::read_parquet("report.parquet"),
output_filename = "report-protein-group.txt",
sample_id = "Run",
intensity_col = "Precursor.Normalised",
intensity_col_sep = NULL,
annotation_col = c("Protein.Ids","Protein.Names", "Genes"),
filter_double_less = c("Q.Value" = "0.01", "PG.Q.Value" = "0.05",
"Lib.Q.Value" = "0.01",
"Lib.PG.Q.Value" = "0.01"))
Similarly, for a DIA-NN search without MBR
iq::process_long_format(arrow::read_parquet("report.parquet"),
output_filename = "report-protein-group.txt",
sample_id = "Run",
intensity_col = "Precursor.Normalised",
intensity_col_sep = NULL,
annotation_col = c("Protein.Ids","Protein.Names", "Genes"),
filter_double_less = c("Q.Value" = "0.01", "PG.Q.Value" = "0.05",
"Global.Q.Value" = "0.01",
"Global.PG.Q.Value" = "0.01"))
Finally, use the parameter peptide_extractor if you want to get the number of peptides per protein, for example with MBR and the --export-quant option.
iq::process_long_format("report.tsv",
output_filename = "report-protein-group.txt",
sample_id = "Run",
intensity_col = "Intensities",
annotation_col = c("Protein.Ids","Protein.Names", "Genes"),
filter_double_less = c("Q.Value" = "0.01", "PG.Q.Value" = "0.05",
"Lib.Q.Value" = "0.01",
"Lib.PG.Q.Value" = "0.01"),
peptide_extractor = function(x) gsub("[0-9].*$", "", x))
To process a Spectronaut output
Use this export schema iq.rs to make a long report, for example "Spectronaut_Report.xls".
process_long_format("Spectronaut_Report.xls",
output_filename = "iq-MaxLFQ.tsv",
sample_id = "R.FileName",
primary_id = "PG.ProteinGroups",
secondary_id = c("EG.Library", "FG.Id", "FG.Charge", "F.FrgIon",
"F.Charge", "F.FrgLossType"),
intensity_col = "F.PeakArea",
annotation_col = c("PG.Genes", "PG.ProteinNames", "PG.FastaFiles"),
filter_string_equal = c("F.ExcludedFromQuantification" = "False"),
filter_double_less = c("PG.Qvalue" = "0.01", "EG.Qvalue" = "0.01"),
log2_intensity_cutoff = 0)