HowDirty is an R package that assesses the level of contamination of LC-MS results. The presence of contaminants (e.g., PEG), and detergents (e.g., CHAPS, SDS) in samples analyzed by LC-MS can be severely detrimental to identifying peptides/proteins or other molecules. Skyline is used to extra MS1 features of many known contaminant masses from raw files (e.g., .raw and .d). The results are exported to a .csv file, then processed in R using HowDirty to generate an HTML interactive report that evaluates sample contamination risks. For more details, please see our publication and the tutorial (below).
Skyline: B. MacLean, D.M. Tomazela, N. Shulman, M. Chambers, G.L. Finney, B. Frewen, R. Kern, D.L. Tabb, D.C. Liebler, M.J. MacCoss, Skyline: an open source document editor for creating and analyzing targeted proteomics experiments, Bioinformatics. 26 (2010) 966–968. [https://doi.org/10.1093/bioinformatics/btq054].
Molecular contaminant transition list: M.J. Rardin, Rapid Assessment of Contaminants and Interferences in Mass Spectrometry Data Using Skyline, J. Am. Soc. Mass Spectrom. 29 (2018) 1327–1330. https://doi.org/10.1007/s13361-018-1940-z.
HowDirty R package: D. Gomez‐Zepeda, T. Michna, T. Ziesmann, U. Distler, S. Tenzer, HowDirty: An R package to evaluate molecular contaminants in LC‐MS experiments, Proteomics. (2023) 1–6. https://doi.org/10.1002/pmic.202300134.
Detailed instructions can be found in the tutorial.
- Raw LC-MS results to be evaluated
- Skyline version > 4 [1,2]. To install it, you can register online and download the latest version here: https://skyline.ms/project/home/software/Skyline/begin.view
- Skyline HowDirty template, including the Skyline molecular contaminant transition list [3] and reports configuration
- Alternatively, you can set up Skyline yourself (further instructions in the Skyline tutorials)
- Download the molecular contaminant transition list [3] from Panorama and load it into Skyline:File / Import / Transition List…
- Create the PeakAreas_Contaminants report: containing the columns:
- Settings / Document Settings / Report / Add, then add a name and select the columns: "Protein", "Peptide", "Replicate Name", "Peptide Retention Time", "Total Area MS1", "Total Ion Current Area"
- Enable the report form by ticking the box next to its name, then click OK
- R software for data analysis and the R packages Rmarkdown, knitr, and HowDirty
install.packages("devtools")
library(devtools)
install_github("kassambara/ggpubr")
install_github("DavidGZ1/HowDirty", force = TRUE)See tutorial for detailed instructions on how to extract MS1 features from Skyline and on pipeline usage.
# Set the working directory to the desired folder (e.g., where the PeakAreas_Contaminants.csv is stored)
setwd("C:/Users/Name/ExampleHowDirty")
# Load HowDirty package
library(HowDirty)
# Create sample annotation or experiment design file
get_annotation_template(file_report_skyline = "PeakAreas_Contaminants.csv")
# Create HowDirty template with the name "example.Rmd"
HowDirty::get_report_template(file = "example")
# Fill the parameters in the header and “knit” (compile) the report. There are two options for this (see screenshots below).
# Using RStudio:
# a) Click on "Knit with parameters"
# b) Fill out the parameters values manually, then click "Knit"
# The parameters are in the header of the .Rmd document after "params:"
# The values are entered after "value: "
# Do not modify the text after "label: " or "input: "Knit button in RStudio:
a)
b)
