Python and R code for peptide identification and quantification from direct infusion shotgun proteome analysis.

https://doi.org/10.26434/chemrxiv.12367376.v1

The code is split into folders for "Python" and "R". The data needed to do targeted and untargeted quantificaiton is under the "data" folder. Outputs from R and Python scripts are in the "outputs" folder.

R is used to:

1. compute peptide-level FDR
2. match peptides to proteins in the FASTA file using the biostrings package. Only peptides that match a single protein entry are kept.
3. generate images of the raw, projected, and library spectra.

Python is used to:

1. fix the MGF spectral library created by msconvert.exe conversion of the ms2 spectral library
2. do the quantification by accessing the raw data using the pyteomics package.

1. fixMGFlib.ipynb - This file contains a few lines of code to add lines of text containing the peptide sequence to the MGF file for use with MSPLIT-DIA
2. DI2A_make_quant_targets_for_R.ipynb - code to process MSPLIT-DIA results and add the heavy and light fragment ion masses to use for quantification. Output is ready for protein-level FDR filtering with R to make the target lists for building targeted instrument methods (make_targetedMS2_lists_for_instrument_method.R) and to make the quantitative list for python targeted Quant (make_quantlist_withMass_for_python.R).
3. DI2A_untargeted_peptide_quantification.ipynb - Performs untargeted peptide quantification
4. DI2A_targeted_protein_quantification.ipynb - Performs targeted peptide quantification using the target list from "make_quantlist_withMass_for_python.R"

1. getSpectra.R - code to generate figures of raw, projected, and library spectra
2. pepFDR.R - function to compute the peptide-level FDR from MSPLIT-DIA output
3. getPepFDRloop.R - code to loop through all the MSPLIT-DIA results in a directory and determine the number of peptides identified at a given FDR
4. make_quantlist_withMass_for_python.R - code to read the python-processed MSPLIT-DIA results (processed with "DI2A_make_quant_targets_file_for_R.ipynb"), do protein FDR filtration, and generate a file containing the quantitative targets for use in python to do targeted peptide quantification (DI2A_targeted_protein_quantification.ipynb).
5. make_targetedMS2_lists_for_instrument_method.R - code to read the python-processed MSPLIT-DIA results (processed with "DI2A_make_quant_targets_file_for_R.ipynb"), do protein FDR filtration, and generate the targeted MS2 lists per CV value needed to generate instrument data collection method on the orbitrap fusion lumos.

1. Table S1 gives the list of 395 proteins identified from the best scouting experiment conditions from MCF7 cells
2. Table S2 gives the MSPLIT-DIA search output from the qualitative analysis of A549 cell samples corresponding to 2,248 unique peptides at <1% peptide-level FDR
3. Table S3 gives the list of 552 peptides targeted to quantify 552 proteins