Code used in MATLAB and R for the purpose of generating HX difference plots and HX ribbon plots
Data files
Jan 22, 2025 version files 21.23 KB
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DiffPlotColors23KK10.m
1.64 KB
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generate_dataframes.R
946 B
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hx-datafiles.csv
130 B
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LUT_ribbon-colors.csv
357 B
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plot_diff_PARP1_HDEx_LZ_012218_5p.m
3.88 KB
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README.md
5.63 KB
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ribbon_maker_functions_2.R
2.20 KB
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ribbon_maker_functions.R
2.20 KB
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ribbon_maker.Rmd
4.25 KB
Abstract
PARP1 and PARP2 recognize DNA breaks immediately upon their formation, generate a burst of local PARylation to signal their location, and are co-targeted by all current FDA-approved forms of PARP inhibitors (PARPi) used in the cancer clinic. Recent evidence indicates that the same PARPi molecules impact PARP2 differently from PARP1, raising the possibility that allosteric activation may also differ. We find that unlike for PARP1, destabilization of the autoinhibitory domain of PARP2 is insufficient for DNA damage-induced catalytic activation. Rather, PARP2 activation requires further unfolding of an active site helix. In contrast, the corresponding helix in PARP1 only transiently forms, even prior to engaging DNA. Only one clinical PARPi, Olaparib, stabilizes the PARP2 active site helix, representing a structural feature with the potential to discriminate small molecule inhibitors. Collectively, our findings reveal unanticipated differences in local structure and changes in activation-coupled backbone dynamics between human PARP1 and PARP2.
README: Code used in MATLAB and R for the purpose of generating HX difference plots and HX ribbon plots
This file was generated on 2024-05-15 by E. Selma Smith
Description of the data and file structure
HDexaminer is a software package used for hydrogen/deuterium exchange mass spectrometry (HXMS) analysis.
HDexaminer takes MS data (.raw) and calculates the deuteration level of measured peptides, which are compared across two protein states. The resulting calculations and measurements sample are exported in a .csv result file. This .csv result file is the input of the ribbon plot (Rstudio) and the difference plot (MATLAB).
Code for ribbon plots (R) was used to define the % deuteration per peptide in triplicate samples of WT PARP2 without or with 5'P nicked DNA, 5'OH nicked DNA, gap DNA, or 5'P nicked DNA and HPF1, N116A PARP2 with or without 5'P nicked DNA, I318A PARP2 (no DNA), and A762 PARP1 with or without 5'P nicked DNA and/or EB-47 (see Smith-Pillet et al., Mol Cell 2025). Each bar represents a peptide within the dataset, colored based on % deuteration.
Ribbon plot code constituents:
- ribbon_maker_functions_2.R --> Contains functions used by ribbon_maker.Rmd that calculate the number of deuterons of the fully deuterated (FD) control peptide.
- ribbon_maker_functions.R--> Contains functions used by ribbon_maker.Rmd that calculate the number of deuterons in the HX sample peptide, relative to the fully deuterated (FD) control peptide.
- LUT_ribbon-colors.csv --> Contains coloring legend scheme for ribbon_maker.Rmd in RGB
- generate_dataframes.R --> contains functions used by ribbon_maker.Rmd to identify peptides based on start, end, and charge state (cs)
- hx-datafiles.csv --> contains directory where filenames of the .csv files exported from HDexaminer should be placed.
- ribbon_maker.Rmd --> Contains code that generates ribbon plot. HX timepoints, plot filetype, plot filename, plot dpi, plot width, plot height, and multiple other formatting details, can be customized in this file.
How to use files (.csv, exported from HDexaminer) in ribbon plot code:
- Create a folder titled "scripts". Folder should include:
- ribbon_maker_functions_2.R --> Contains functions used by ribbon_maker.Rmd
- ribbon_maker_functions.R--> Contains functions used by ribbon_maker.Rmd
- LUT_ribbon-colors.csv --> Contains coloring legend scheme
- generate_dataframes.R --> contains functions used by ribbon_maker.Rmd
- In folder titled "HDExaminer_csv". Folder should include:
- .Rhistory
- .csv files (exported from HDexaminer) of non-deuterated (ND), fully-deuterated (FD), and partially deuterated samples (aka on-exchange).
- hx-datafiles.csv
- ribbon_maker.hmtl
- ribbon_maker.Rmd
- Add titles of .csv files in the hx-datafiles.csv file, containing ND, FD, and on-exchange samples per condition.
- Add .csv files (exported from HD examiner) to "HDExaminer_csv" folder.
Note: All .csv files cannot have peptides with low-confidence. All .csv files must have the same peptides listed in each row. This is best performed ahead of time in HDexaminer.
5. Customize colors corresponding to % HX values in LUT_ribbon-colors.csv. Color values are formatted in RGB.
6. Open "ribbon_maker.Rmd" in Rstudio and run all.
Code for difference plots (MATLAB R2017A) was used to define the difference in % deuteration per peptide in triplicate samples of WT or mutant PARP1 or PARP2 under the conditions indicated in the figure legends.
This code will prompt the selection of two .csv files (exported from HDexaminer) that are compared.
Each bar represents the difference in deuteration per peptide, colored based on the difference in % deuteration between the sample conditions. The color legend indicating the severity of differences in % deuteration is determined by the "DiffPlotColors" code. Colors are RGB format.
Constituents of HX difference plot code:
- DiffPlotColors23KK10.m --> contains color scheme of peptide %HX ranges in RGB (R,G,B) on a scale from 0 to 1.
- plot_diff_PARP1_HDEx_LZ_012218.m --> contains code that is run to generate a HX difference plot comparing a single protein across two different conditions (e.g., salt concentrations or +/- DNA).
How to use deposited files utilized to create difference plots (MATLAB):
- In one folder, download "DiffPlotColors23KK10.m" and "plot_diff_PARP1_HDEx_LZ_012218.m"
- Customize colors associated with each % difference in HX in "DiffPlotColors23KK10.m"
- Open "plot_diff_PARP1_HDEx_LZ_012218.m" and Run. This code is used to generate the HX difference plot.
- User will be prompted to select two .csv files (two on-exchange samples being compared, exported from HDexaminer). Code will plot the HX difference onto colored rectangles by subtracting the %HX of the 2nd .csv file from the first.
Sharing/Access information
SHARING/ACCESS INFORMATION
1. Licenses/restrictions placed on the data: None
2. Links to publications that cite or use the data: Will update before publication
3. Links to other publicly accessible locations of the data: None
4. Links/relationships to ancillary data sets: None
5. Was data derived from another source? No
6. Recommended citation for this dataset: n/a
Code/Software
Version of Rstudio used:
- RStudio 2021.09.1+372 "Ghost Orchid" Release (8b9ced188245155642d024aa3630363df611088a, 2021-11-08) for Windows
- Mozilla/5.0 (Windows NT 6.1; Win64; x64) AppleWebKit/537.36 (KHTML, like Gecko) QtWebEngine/5.12.8 Chrome/69.0.3497.128 Safari/537.36
Version of MATLAB used:
- R2017a (9.2.0.556344)
Methods
HDExaminer software (v 2.5.0) was used, which uses peptide pool information to identify the deuterated peptides for every sample in the HXMS experiment. The quality of each peptide was further assessed by manually checking mass spectra. The level of HX of each reported deuterated peptide is corrected for loss of deuterium label (back-exchange after quench) during HXMS data collection by normalizing to the maximal deuteration level of that peptide in the fully-deuterated (FD) samples. After normalizing, we then compared the extent of deuteration to the theoretical maximal deuteration (maxD, i.e. if no back-exchange occurs). The data analysis statistics for all the protein states are in Table S2 of Smith-Pillet et al., Mol cell 2025. The difference plots for the deuteration levels between any two samples were obtained through an in-house script written in MATLAB. The script compares the deuteration levels between two samples (e.g. PARP2 and PARP2 with 5’P nicked DNA) and plots the percent difference of each peptide, by subtracting the percent deuteration of PARP2 with 5’P nicked DNA from PARP2 and plotting according to the color legend in stepwise increments. The plot of representative peptide data is shown as the mean of three independent measurements +/- SD. Statistical analysis included a t-test with a P-value <0.05. HX experiments of PARP1 with or without DNA and/or EB-47 have been published. To compare PARP1 and PARP2 datasets, HX samples of PARP1 were repeated in triplicate to have the same peptide digestions and subsequent peptide data, and HX changes in HD peptides were compared between PARP1 and PARP2 with the indicated conditions. HXMS data at 100 s for PARP2 and in the presence of gap DNA, 5’OH nicked DNA, and 5’P nicked DNA was plotted through an in-house script written in R (see Fig. S1A in Smith-Pillet et al., Mol cell 2025).