iq: an R package for protein quantification

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

Installation

The package is hosted on CRAN. It is best to install from within R.

install.packages("iq")

Usage

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::processlongformat("report.tsv", outputfilename = "report-protein-group.txt", sampleid = "Run", intensitycol = "Intensities", annotationcol = c("Protein.Ids","Protein.Names", "Genes"), filterdoubleless = 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)