Parse Freestyle Excel outputs

Required inputs:

• raw_data: path to a folder containing multiple Excel files (e.g. Project_01/Freestyle_outputs), or path pointing to a single Excel file (e.g. Project_01/Freestyle_outputs/Outputfile@IgM_site4.xlsx). Note that the target peptide name has to be appended unambiguously to the filename of each Excel file (e.g. @peptide_X.xls)

• config_file: path to a 2-columns .csv file containing:

  • 1st row - tolerance_value followed by the value (in ppm) setting the stringency of peaks' annotation
  • 2nd row - overlap_label_range followed by the value (in Dalton) defining a range within which only the highest intensity peak will be labbeled in the plot
  • 3nd row - mass_range followed by the values' range (in Dalton:Dalton) of min and max Monoisotopic mass that will be plotted. Use NA to allows for automatic selection of the value/s (e.g. 750:NA)
  • 4nd row - modification_name followed by the string defining the name of expected modification (e.g., NH4_adduct, Formyl) that create the adducts. Use NA to skip modification check
  • 5nd row - modification_mass followed by the values (in Dalton) of the expected ions causing the adducts (e.g., 18.03382 for NH4^+^, 27.994915 for CHO). If modification_name is set to NA, this row will be disregarded. Use negative value to indicate loss of such mass (e.g., -18.03382 for loss of NH4^+^)
  • n-rows containing names of any analyzed peptides followed by their masses (in Dalton)

  File example:

   tolerance_value,5
   overlap_label_range,50
   mass_range,NA:NA
   modification_name,Formyl
   modification_mass,27.994915
   IgG,1189.512
   IgM_site1,1284.618
   IgM_site4,1216.559
   Aflibercept_site1,1172.6885    
  

• annotation_db: path to a 2-columns .csv file containing the list of glycoform names and related glycan masses. Note that the first row in the file contain the column names. For the glycoform names, use '_' instead of spaces, and '@' to separate name types, e.g. "H5N2@Man5"

  File example:

   glycoform,Glycan_Mass
   N1@single-GlcNAc_(Gn),203.0794
   H2N2@MU_,730.2644
   H2N2X@MUX_,862.3067
   H3N2@MM_,892.3172
  

• out_dir: path to a folder in which the output files will be stored. NB the script will overwirte any file with standard names (i.e. Peaks_annotated_all.txt/.pdf and Peaks_annotated_clean.txt/.pdf) in this path.

Set-up the environment:

• R needs to be installed on the computer. Please follow the instruction provided under Download and Install R at https://cran.r-project.org/

• Install the necessary packages:

  • Start an R session

  • Copy and paste the following code in the R console and follow the prompted instructions:

    required_libs <- c("optparse","readxl","tidyverse","ggplot2","ggrepel")
    missing_libs <- required_libs[!required_libs %in% rownames(installed.packages())]
    if (length(missing_libs) > 1) {
      cat("Installing missing packages: ", paste(missing_libs, collapse = ", "), "\n\n")
      sapply(missing_libs, function(i) install.packages(i))
    }
    invisible(lapply(required_libs, library, character.only = TRUE))
    

• Fetch the tool repository

  • in Windows:

    • Almost at the top of this GitHub page, click "<> Code"
    • Click on "Download ZIP"
    • Save and extract the file where you prefer (e.g. C:\Users\Luca\Tools)

  • in Linux:

    open a terminal and type:

    sudo apt update
    sudo apt install git
    cd /home/user/tools # or to whatever folder in which you want to save the tool
    git clone https://github.com/lucaz88/Freestyle_parser.git
    

How to run:

For Windows system:

!!! Edit paths to point to the where you have actually installed R and downloaded the repo

• Press the Windows Start button on the screen or keyboard

• Type in "Command Prompt"

• Left click on Command Prompt

• Move into the repository folder cd C:\Users\Luca\Tools\Freestyle_parser-main in which toy files are provided to test the tool

• Type "C:\Program Files\R\R-4.2.2\bin\Rscript.exe" Freestyle_parser.R -r toy_data -a annotation_db.csv -c config_file.csv -o res_toy

• To test the adducts option type "C:\Program Files\R\R-4.2.2\bin\Rscript.exe" Rscript Freestyle_parser.R -r toy_adducts -a annotation_db.csv -c config_file_adducts.csv -o res_adducts

For Linux system:

open a terminal and type:

cd /home/user/tools/Freestyle_parser
Rscript Freestyle_parser.R -r toy_data -a annotation_db.csv -c config_file.csv -o res_toy

# To test the adducts option:
Rscript Freestyle_parser.R -r toy_adducts -a annotation_db.csv -c config_file_adducts.csv -o res_adducts

To run you own sample simply edit the paths of the arguments -r, -a, -c and -o so that they point to the relevant folder/files. Otherwise, cd into your project folders, containing the needed files, and add the full path to the script file in the command (e.g. Rscript /home/user/tools/Freestyle_parser/Freestyle_parser.R -r ...).