Data from: Phosphoproteome-derived peptide libraries for deep specificity profiling of phosphatases and phospholyases

Radziwon, Katarzyna1; Campbell, Laura1; Mazurkiewicz, Lauren1; Jalalishvili, Sopo1; Eppinger, Izabelle1; Parikh, Aanika1; Weeks, Amy 1

Published May 22, 2026 on Dryad. https://doi.org/10.5061/dryad.95x69p8z0

Abstract

Protein phosphorylation is dynamically regulated by phosphowriter enzymes (kinases) and phosphoeraser enzymes (phosphatases and phospholyases). While kinase sequence preferences have been extensively characterized, phosphoeraser selectivity has not been systematically explored. This dataset supports the development of an experimental platform based on tandem mass spectrometry analysis of phosphoproteome-derived peptide libraries (PhosPropels) to profile phosphoeraser activity across thousands of biologically relevant phosphosites. Using this approach, we defined positional residue preferences for eight phosphoerasers spanning diverse species, protein folds, and catalytic mechanisms. We further profiled 20 variants of the phosphothreonine lyase OspF from Shigella flexneri, revealing intrinsic selectivity for p38 and Erk MAP kinase activation loops and identifying enzyme residues that influence phosphothreonine specificity.

This deposition includes: Plasmid maps for expression constructs of OspF, OspF (27–239), WipA, and WipB; quantitative proteomics summary datasets and associated raw data; uncropped Western blots used for analysis of inducible OspF activity in cells; raw .nd2 fluorescence microscopy images; and AlphaFold3 structural models and accompanying files for WipA and WipB. Together, these materials support reproducibility of the experiments and enable reuse of the datasets and structural models for further analysis of phosphoeraser specificity.

Dataset DOI: 10.5061/dryad.95x69p8z0

Description of the data and file structure

These data and files were collected to enable the development of phosphoproteome-derived peptide libraries as a tool for phosphoeraser specificity profiling.

Files and variables

File: Plasmid_maps.zip

Description: This folder contains the following plasmid maps:

pbh4_ospf.gb. The E. coli codon-optimized gene encoding OspF was inserted into the pBH4 vector between the BamHI and NotI restriction sites using Gibson assembly, fusing OspF with an N-terminal His tag followed by a TEV protease cleavage site.
pbh4_ospf27-39.gb: The E. coli codon-optimized gene encoding OspF(27-239) was inserted into the pBH4 vector between the BamHI and NotI restriction sites using Gibson assembly, fusing OspF(27-239) with an N-terminal His tag followed by a TEV protease cleavage site.
pet28a_wipa.gb: The E. coli codon-optimized gene encoding WipA was inserted into the pET28a vector between the NcoI and NotI restriction sites using Gibson assembly to generate a construct with a C-terminal His tag.
pbh4_wipb.gb: The E. coli codon-optimized gene encoding WipB1-364 was inserted into the pBH4 vector between the BamHI and NotI restriction sites using Gibson assembly to fuse WipB with an N-terminal His tag followed by a TEV protease cleavage site.
pcdna5frtto-ospf.gb: The human codon-optimized gene encoding V5-OspF was inserted into the pcDNA5/FRT/TO vector (Invitrogen) between the KpnI and NotI restriction sites using Gibson assembly.

File: Fluorescence_microscopy_data.zip

Description: This folder contains unprocessed .nd2 files that are fluorescence microscopy images of HEK293T cells expressing V5-tagged OspF under a doxycycline-inducible promoter. Anti-V5 is in the red channel, and DAPI is in the blue channel.

ospf_flpin_no_dox_control_well1_008.nd2: Uninduced cells
ospf_flpin_plus_dox_induced_well4_004.nd2: Induced cells

File: Uncropped_Western_blots.zip

Description: This folder contains uncropped Western blot .tif files for blots of pErk, pp-p38, and ppJNK and appropriate loading controls. Blots were performed as described in the text using LiCOR imaging.

ospf_flpin_no_dox_control_well1_008.nd2: Uninduced cells
ospf_flpin_plus_dox_induced_well4_004.nd2: Induced cells

Data collection and analysis:

FlpIn-HEK293T cells expressing OspF or OspF-K134A under a doxycycline-inducible promoter (untreated or stimulated with EGF or anisomycin) were treated with doxycycline to induce OspF expression, or a vehicle control
Western blotting was then performed with antibodies to dual-phosphorylated Erk, p38, or JNK
Western blots were imaged using LiCOR imaging

How the data are organized:

README.txt — This file

Subdirectories - replicate datasets for each timepoint are arranged into the following subdirectories:

├── pp-p38_EGF_treated
├── pp-p38_untreated
├── ppErk_EGF_treated
├── ppErk_untreated
├── ppJNK_anisomycin_treated
└── ppJNK_untreated

Each subdirectory contains a PDF file (*_layout.pdf) with an annotated blot image that describes which samples were in which lanes and which uncropped images correspond to which experiments; a tif file named *-[IRshort].tif that corresponds to the 680 nm imaging channel used for the phosphophorylated kinase; and a tif file name *-[IRlong].tif that corresponds to the 800 nm imaging channel used for the loading control.

File: Supplementary_Dataset_1_-_HEK293T_untreated_phosphopeptide_library.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for 7 replicate phosphoproteome-derived peptide libraries made from untreated HEK293T cells.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using TiO2 enrichment as described in the accompanying manuscript.
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

HEK293T_untreated_rep1_E20211231-03_ptmRS_PeptideGroups.csv
HEK293T_untreated_rep2_E20211231-05_ptmRS_PeptideGroups.csv
HEK293T_untreated_rep3_E20211231-07_ptmRS_PeptideGroups.csv
HEK293T_untreated_rep4_E20211231-09_ptmRS_PeptideGroups.csv
HEK293T_untreated_rep5_E20211231-11_ptmRS_PeptideGroups.csv
HEK293T_untreated_rep6_E20220519-06_ptmRS_PeptideGroups.csv
HEK293T_untreated_rep7_E20220519-08_ptmRS_PeptideGroups.csv

Datafile names contain a description (e.g., HEK293T_untreated_rep1 corresponding to a phosphoproteome-derived peptide library from untreated HEK293T cells, replicate 1), followed by the name of the file (e.g., E20211231-03, which can be connected to specific peak, raw, and result files in our ProteomeXchange deposition), followed by a description of the result type (e.g., ptmRS_PeptideGroups, corresponding to Peptide Groups that have been assigned site localization scores using ptmRS).

Each file contains a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_2_-_HEK293T_pervanadate-treated_phosphopeptide_library.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for 4 replicate phosphoproteome-derived peptide libraries made from pervanadate-treated HEK293T cells.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using TiO2 enrichment as described in the accompanying manuscript.
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

HEK293T_pervanadate_rep1_E20231207-03_ptmRS_PeptideGroups.csv
HEK293T_pervanadate_rep2_E20240823-08_ptmRS_PeptideGroups.csv
HEK293T_pervanadate_rep3_E20240907-04_ptmRS_PeptideGroups.csv
HEK293T_pervanadate_rep4_E20241205-04_ptmRS_PeptideGroups.csv

Datafile names contain a description (e.g., HEK293T_pervanadate_rep1 corresponding to a phosphoproteome-derived peptide library from pervanadate-treated HEK293T cells, replicate 1), followed by the name of the file (e.g., E20231207-03, which can be connected to specific peak, raw, and result files in our ProteomeXchange deposition), followed by a description of the result type (e.g., ptmRS_PeptideGroups, corresponding to Peptide Groups that have been assigned site localization scores using ptmRS).

Each file contains a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_2_-_HEK293T_pervanadate-treated_phosphopeptide_library.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for 4 replicate phosphoproteome-derived peptide libraries made from pervanadate-treated HEK293T cells.

Data collection and analysis:

Phosphoproteome-derived peptide libaries were prepared using TiO2 enrichment as described in the accompanying manuscript.
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

HEK293T_pervanadate_rep1_E20231207-03_ptmRS_PeptideGroups.csv
HEK293T_pervanadate_rep2_E20240823-08_ptmRS_PeptideGroups.csv
HEK293T_pervanadate_rep3_E20240907-04_ptmRS_PeptideGroups.csv
HEK293T_pervanadate_rep4_E20241205-04_ptmRS_PeptideGroups.csv

Each file contains a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_3_-_Jurkat_pervanadate-treated_phosphopeptide_library.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for 3 replicate phosphoproteome-derived peptide libraries made from pervanadate-treated Jurkat cells.

Data collection and analysis:

Phosphoproteome-derived peptide libaries were prepared using TiO2 enrichment as described in the accompanying manuscript.
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

Jurkat_pervanadate_rep1_E20250513-12_PeptideGroups.csv
Jurkat_pervanadate_rep2_E20250513-14_PeptideGroups.csv
Jurkat_pervanadate_rep3_E20250513-16_PeptideGroups.csv

Datafile names contain a description (e.g., Jurkat_pervanadate_rep1 corresponding to a phosphoproteome-derived peptide library from pervanadate-treated Jurkat cells, replicate 1), followed by the name of the file (e.g., E20250513-12, which can be connected to specific peak, raw, and result files in our ProteomeXchange deposition), followed by a description of the result type (e.g., _PeptideGroups, corresponding to Peptide Groups that have been assigned site localization scores using ptmRS).

Each file contains a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_4_-_K562_pervanadate-treated_phosphopeptide_library.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for 4 replicate phosphoproteome-derived peptide libraries made from pervanadate-treated K562 cells.

Data collection and analysis:

Phosphoproteome-derived peptide libaries were prepared using TiO2 enrichment as described in the accompanying manuscript.
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

K562_pervanadate_rep1_E20250523-06_PeptideGroups.csv
K562_pervanadate_rep2_E20250523-08_PeptideGroups.csv
K562_pervanadate_rep3_E20250523-10_PeptideGroups.csv
K562_pervanadate_rep4_E20250523-12_PeptideGroups.csv

Datafile names contain a description (e.g., K562_pervanadate_rep1 corresponding to a phosphoproteome-derived peptide library from pervanadate-treated K562 cells, replicate 1), followed by the name of the file (e.g., E20250523-06, which can be connected to specific peak, raw, and result files in our ProteomeXchange deposition), followed by a description of the result type (e.g., _PeptideGroups, corresponding to Peptide Groups that have been assigned site localization scores using ptmRS).

Each file contains a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_5_-_Average_kinase_substrate_counts.zip

Description: Each sheet in this Excel file corresponds to one type of PhosPropel which is indicated in the name of the tab. Within each sheet, there are two columns: Kinase, which contains the HGNC-format gene name for each kinase, and Average Substrate Count. The Average Substrate Count column indicates the average number of times a phosphosite annotated as a potential substrate of a particular kinase was observed across replicate datasets. To annotate substrates, we scored each phosphosite using PSSMs reported in refs 1 and 2. Potential substrates were those with a PSSM score >= 2 s.d. above the mean score for that kinase.

References

Johnson, J.L. et al. An atlas of substrate specificities for the human serine/threonine kinome. Nature 613, 759-766 (2023).
Yaron-Barir, T.M. et al. The intrinsic substrate specificity of the human tyrosine kinome. Nature 629, 1174-1181 (2024).

File: Supplementary_Dataset_6_-_HEK293T_pervanadate-treated_phosphopeptide_library_treated_with_lambda_phosphatase.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with lambda phosphatase and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libaries were prepared using TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with lambda phosphatase as described in the manuscript and quenched at different time points
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Subdirectories - replicate datasets for each timepoint are arranged into the following subdirectories:

├── 0min/
├── 5min/
├── 30min/
└── 180min/

Each folder contains csv files corresponding to the Peptide Groups tab of search results from Proteome Discoverer. Each csv file is one replicate for the timepoint named in the subdirectory name. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_7_-_HEK293T_pervandate-treated_phosphopeptide_library_treated_with_PTP1B_catalytic_domain.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with PTP1B(1-321) and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libaries were prepared using TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with lambda phosphatase as described in the manuscript and quenched at different time points
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Subdirectories - replicate datasets for each timepoint are arranged into the following subdirectories:

├── 0min/
├── 5min/
├── 30min/
└── 60min/

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_8_-_HEK293T_pervanadate-treated_phosphopeptide_library_treated_with_PP2Ac.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with the catalytic subunit of PP2A (PP2Ac) and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libaries were prepared using TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with lambda phosphatase as described in the manuscript and quenched at different time points
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Subdirectories - replicate datasets for each timepoint are arranged into the following subdirectories:

├── 0min/
├── 5min/
├── 30min/
└── 180min/

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_9_-_HEK293T_pervanadate-treated_library_treated_with_WipA.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with WipA and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with lambda phosphatase as described in the manuscript and quenched at different time points
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Subdirectories - replicate datasets for each timepoint are arranged into the following subdirectories:

├── 0min/
├── 5min/
├── 30min/
└── 120min/

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_10_-_HEK293T_pervanadate-treated_library_treated_with_WipB.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with WipB and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with lambda phosphatase as described in the manuscript and quenched at different time points
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Subdirectories - replicate datasets for each timepoint are arranged into the following subdirectories:

├── 0min/
├── 5min/
├── 30min/
└── 120min/

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_11_-_HEK293T_pervanadate-treated_library_treated_with_OspF.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with OspF.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with OspF as described in the manuscript and quenched at different time points
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Data files:

osp_HEK293T_pervanadate_rep3_E20241204-05_ptmRS_PeptideGroups.csv
ospf_HEK293T_pervanadate_rep1_E20241204-01_ptmRS_PeptideGroups.csv
ospf_HEK293T_pervanadate_rep2_E20241204-03_ptmRS_PeptideGroups.csv

Each csv file is one replicate. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_12_-_HEK293T_pervanadate-treated_library_treated_with_SpvC_or_HopAI.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with phospholyases SpvC and HopAI.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with lambda phosphatase as described in the manuscript and quenched at different time points
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Subdirectories - replicate datasets for each timepoint are arranged into the following subdirectories:

├── spvc_perlib_ptmrs/
└── hopai_perlib_ptmrs/

Each folder contains csv files corresponding to the Peptide Groups tab of search results from Proteome Discoverer. Each csv file is one replicate for the experiment in the subdirectory name. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_13_-_HEK293T_pervanadate-treated_library_treated_with_OspF_27-239.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with OspF (residues 27-239).

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with OspF (residues 27-239) as described in the manuscript and quenched at different time points
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Data files:

ospf_delN_rep1_E20250326-06_ptmrs_PeptideGroups.csv
ospf_delN_rep2_E20250326-10_ptmrs_PeptideGroups.csv

Each csv file is one replicate. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_14_-_OspF_TMT_results.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for a TMT phosphoproteomics experiment in compared HEK293T cells in which OspF expression has been induced to uninduced cells.

Data collection and analysis:

Phospho-enriched TMT samples were prepared as described in the accompanying manuscript.
LC-MS/MS data were collected on an Orbitrap Lumos mass spectrometer (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Files:

OspF_TMT_results_peptides.csv/ --> peptide identification and quantification
OspF_TMT_results_proteins.csv/ --> protein identification and quantification

OspF_TMT_results_peptides.csv corresponds to the Peptide Groups tab of search results from Proteome Discoverer. The columns in the csv are:

Checked
Confidence
Annotated Sequence
Modifications
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions
Positions in Master Proteins
Modifications in Master Proteins
# Missed Cleavages
Theo. MH+ [Da]
Abundance Ratio: (induced) / (control)
Abundance Ratio Adj. P-Value: (induced) / (control)
Abundances (Grouped): control
Abundances (Grouped): induced
Abundances (Grouped) CV [%]: control
Abundances (Grouped) CV [%]: induced
Quan Info
Found in Sample: [S1] F1: 126, Sample, control
Found in Sample: [S2] F1: 127N, Sample, control
Found in Sample: [S3] F1: 127C, Sample, control
Found in Sample: [S4] F1: 128N, Sample, control
Found in Sample: [S5] F1: 128C, Sample, induced
Found in Sample: [S6] F1: 129N, Sample, induced
Found in Sample: [S7] F1: 129C, Sample, induced
Found in Sample: [S8] F1: 130N, Sample, induced
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

OspF_TMT_results_proteins.csv corresponds to the Protein Groups tab of search results from Proteome Discoverer. The columns in the csv are:

Checked
Protein FDR Confidence: Combined
Master
Accession
Description
Exp. q-value: Combined
Sum PEP Score
Coverage [%]
# Peptides
# PSMs
# Unique Peptides
# AAs
MW [kDa]
calc. pI
Score Sequest HT: Sequest HT
# Peptides (by Search Engine): Sequest HT
# Protein Pathway Groups
# Razor Peptides
Abundance Ratio: (induced) / (control)
Abundance Ratio Adj. P-Value: (induced) / (control)
Abundances (Grouped): control
Abundances (Grouped): induced
Abundances (Grouped) CV [%]: control
Abundances (Grouped) CV [%]: induced
Found in Sample: [S1] F1: 126, Sample, control
Found in Sample: [S2] F1: 127N, Sample, control
Found in Sample: [S3] F1: 127C, Sample, control
Found in Sample: [S4] F1: 128N, Sample, control
Found in Sample: [S5] F1: 128C, Sample, induced
Found in Sample: [S6] F1: 129N, Sample, induced
Found in Sample: [S7] F1: 129C, Sample, induced
Found in Sample: [S8] F1: 130N, Sample, induced
# Protein Groups
Modifications

File: Supplementary_Dataset_15_-_OspF_alanine_variant_activity_on_HEK293T_phosphopeptide_library.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with OspF variants.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with lambda phosphatase as described in the manuscript and quenched at different time points
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Subdirectories - replicate datasets for each variant are arranged into subdirectories named for the variant.

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HTFile

File: Supplementary_Dataset_16_-_Additional_OspF_variant_activity_on_HEK293T_phosphopeptide_library.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with OspF variants.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with lambda phosphatase as described in the manuscript and quenched at different time points
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Subdirectories - replicate datasets for each variant are arranged into the following subdirectories:

├── ospf_d219i_plib_ptmrs/
├── ospf_e213q_plib_ptmrs/
└── ospf_e213q_d219i_plib_ptmrs/

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_17_-_HEK293T_pervanadate-treated_phosphopeptide_library_GluC.zip

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using GluC digestion followed by TiO2 enrichment as described in the accompanying manuscript.
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

HEK293T_pervanadate_GluC_rep1_E20251021-06.csv
HEK293T_pervanadate_GluC_rep2_E20251021-10.csv
HEK293T_pervanadate_GluC_rep3_E20251021-14.csv
HEK293T_pervanadate_GluC_rep4_E20251021-18.csv
HEK293T_pervanadate_GluC_rep5_E20251030-02.csv
HEK293T_pervanadate_GluC_rep6_E20251113-06.csv
HEK293T_pervanadate_GluC_rep7_E20251113-08.csv

Each file contains a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_18_-_HEK293T_pervanadate-treated_phosphopeptide_library_LysC.zip

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using LysC digestion followed by TiO2 enrichment as described in the accompanying manuscript.
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

HEK293T_pervanadate_LysC_rep1_E20251021-04.csv
HEK293T_pervanadate_LysC_rep2_E20251021-08.csv
HEK293T_pervanadate_LysC_rep3_E20251021-12.csv
HEK293T_pervanadate_LysC_rep4_E20251021-16.csv
HEK293T_pervanadate_LysC_rep5_E20251113-04.csv

Each file contains a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_19_-_HEK293T_pervanadate-treated_phosphopeptide_library_chymotrypsin.zip

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using chymotrypsin digestion followed by TiO2 enrichment as described in the accompanying manuscript.
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

HEK293T_pervanadate_chymotrypsin_rep1_E20260204-04.csv
HEK293T_pervanadate_chymotrypsin_rep2_E20260204-06.csv
HEK293T_pervanadate_chymotrypsin_rep3_E20260204-08.csv
HEK293T_pervanadate_chymotrypsin_rep4_E20260204-10.csv

Each file contains a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_20_-_HEK293T_pervanadate-treated_GluC_phosphopeptide_library_treated_with_lambda_phosphatase.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with lambda phosphatase and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using GluC digestion followed by TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with lambda phosphatase as described in the manuscript and quenched at 3 h (180 min)
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

lambda_pp_180min_GluC_ctrl_E20251118-24.csv - no enzyme control
lambda_pp_180min_GluC_rep1_E20251118-26.csv
lambda_pp_180min_GluC_rep2_E20251118-28.csv
lambda_pp_180min_GluC_rep3_E20251118-30.csv

Each datafile is a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. Each csv file is one replicate. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_21_-_HEK293T_pervanadate-treated_GluC_phosphopeptide_library_treated_with_PP2Ac.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with lambda phosphatase and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using GluC digestion followed by TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with PP2Ac as described in the manuscript and quenched at 3 h (180 min)
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

pp2ac_180min_GluC_ctrl_E20251202-06.csv - no enzyme control
pp2ac_180min_GluC_rep1_E20251202-08.csv
pp2ac_180min_GluC_rep2_E20251202-10.csv
pp2ac_180min_GluC_rep3_E20251202-12.csv

Each datafile is a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. Each csv file is one replicate. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_22_-_HEK293T_pervanadate-treated_GluC_phosphopeptide_library_treated_with_PTP1B.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with lambda phosphatase and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libaries were prepared using GluC digestion followed by TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with PP2Ac as described in the manuscript and quenched at 1 h (60 min)
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

ptp1b_60min_GluC_rep1_E20260106-06.csv
ptp1b_60min_GluC_rep2_E20260106-08.csv
ptp1b_60min_GluC_rep3_E20260106-10.csv

Each datafile is a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. Each csv file is one replicate. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_23_-_HEK293T_pervanadate-treated_GluC_phosphopeptide_library_treated_with_WipA.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with lambda phosphatase and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using GluC digestion followed by TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with WipA as described in the manuscript and quenched at 2 h (120 min)
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

wipa_120min_GluC_ctrl_E20251128-06.csv - no enzyme control
wipa_120min_GluC_rep1_E20251128-08.csv
wipa_120min_GluC_rep2_E20251128-10.csv
wipa_120min_GluC_rep3_E20251128-12.csv

Each datafile is a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. Each csv file is one replicate. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_24_-_HEK293T_pervanadate-treated_GluC_phosphopeptide_library_treated_with_WipB.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with lambda phosphatase and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using GluC digestion followed by TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with WipA as described in the manuscript and quenched at 2 h (120 min)
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

wipa_120min_GluC_ctrl_E20251128-06.csv - no enzyme control
wipa_120min_GluC_rep1_E20251128-08.csv
wipa_120min_GluC_rep2_E20251128-10.csv
wipa_120min_GluC_rep3_E20251128-12.csv

Each datafile is a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. Each csv file is one replicate. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_25_-_HEK293T_pervanadate-treated_GluC_phosphopeptide_library_treated_with_OspF.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with lambda phosphatase and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using GluC digestion followed by TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with OspF as described in the manuscript and quenched at 24 h
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

ospf_24h_GluC_ctrl_E20260106-12.csv - no enzyme control
ospf_24h_GluC_rep1_E20260106-14.csv
ospf_24h_GluC_rep2_E20260106-16.csv
ospf_24h_GluC_rep3_E20260106-18.csv

Each datafile is a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. Each csv file is one replicate. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_26_-_HEK293T_pervanadate-treated_GluC_phosphopeptide_library_treated_with_OspF27-239.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with lambda phosphatase and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using GluC digestion followed by TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with OspF(27-239) as described in the manuscript and quenched at 24 h
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

ospf_27-239_24h_GluC_ctrl_E20260106-12.csv - no enzyme control
ospf_27-239_24h_GluC_rep1_E20260106-20.csv
ospf_27-239_24h_GluC_rep2_E20260106-22.csv
ospf_27-239_24h_GluC_rep3_E20260106-24.csv

Each datafile is a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. Each csv file is one replicate. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_27_-_HEK293T_pervanadate-treated_LysC_phosphopeptide_library_treated_with_lambda_phosphatase.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with lambda phosphatase and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using LysC digestion followed by TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with lambda phosphatase as described in the manuscript and quenched at 3 h (180 min)
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

lambda_pp_180min_LysC_ctrl_E20251113-10.csv - no enzyme control
lambda_pp_180min_LysC_ctrl2_E20251128-18.csv - no enzyme control
lambda_pp_180min_LysC_rep1_E20251128-14.csv
lambda_pp_180min_LysC_rep2_E20251128-16.csv
lambda_pp_180min_LysC_rep3_E20251128-20.csv

Each datafile is a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. Each csv file is one replicate. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_28_-_HEK293T_pervanadate-treated_LysC_phosphopeptide_library_treated_with_PP2Ac.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with lambda phosphatase and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using LysC digestion followed by TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with PP2Ac as described in the manuscript and quenched at 3 h (180 min)
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

pp2ac_180min_LysC_ctrl_E20251111-22.csv - no enzyme control
pp2ac_180min_LysC_ctrl2_E20251204-06.csv - no enzyme control
pp2ac_180min_LysC_rep1_E20251111-24.csv
pp2ac_180min_LysC_rep2_E20251111-26.csv
pp2ac_180min_LysC_rep3_E20251204-08.csv

Each datafile is a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. Each csv file is one replicate. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_29_-_HEK293T_pervanadate-treated_LysC_phosphopeptide_library_treated_with_PTP1B.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with lambda phosphatase and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using LysC digestion followed by TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with PTP1B as described in the manuscript and quenched at 1 h (60 min)
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

ptp1b_60min_LysC_ctrl_E20251217-04.csv - no enzyme control
ptp1b_60min_LysC_rep1_E20251217-06.csv
ptp1b_60min_LysC_rep2_E20251218-06.csv
ptp1b_60min_LysC_rep3_E20251218-08.csv

Each datafile is a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. Each csv file is one replicate. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_30_-_HEK293T_pervanadate-treated_LysC_phosphopeptide_library_treated_with_WipA.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with lambda phosphatase and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using LysC digestion followed by TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with WipA as described in the manuscript and quenched at 2 h (120 min)
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

wipa_120min_LysC_ctrl_E20251024-06.csv - no enzyme control
wipa_120min_LysC_rep1_E20251024-08.csv
wipa_120min_LysC_rep2_E20251024-10.csv
wipa_120min_LysC_rep3_E20251024-12.csv

Each datafile is a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. Each csv file is one replicate. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_31_-_HEK293T_pervanadate-treated_LysC_phosphopeptide_library_treated_with_WipB.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with lambda phosphatase and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using LysC digestion followed by TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with WipB as described in the manuscript and quenched at 2 h (120 min)
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

wipb_120min_LysC_ctrl_E20251024-04.csv - no enzyme control
wipb_120min_LysC_rep1_E20251024-14.csv
wipb_120min_LysC_rep2_E20251024-16.csv
wipb_120min_LysC_rep3_E20251024-18.csv

Each datafile is a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. Each csv file is one replicate. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_32_-_HEK293T_pervanadate-treated_LysC_phosphopeptide_library_treated_with_OspF.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with lambda phosphatase and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using LysC digestion followed by TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with OspF as described in the manuscript and quenched at 24 h
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

ospf_24h_LysC_ctrl_E20251107-04.csv - no enzyme control
ospf_24h_LysC_rep1_E20251107-06.csv
ospf_24h_LysC_rep2_E20251107-08.csv
ospf_24h_LysC_rep3_E20251107-10.csv

Each datafile is a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. Each csv file is one replicate. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_33_-_HEK293T_pervanadate-treated_LysC_phosphopeptide_library_treated_with_OspF27-239.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with lambda phosphatase and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using LysC digestion followed by TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with OspF(27-239) as described in the manuscript and quenched at 24 h
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

ospf_27-239_24h_LysC_ctrl_E20251107-04.csv - no enzyme control
ospf_27-239_24h_LysC_rep1_E20251107-12.csv
ospf_27-239_24h_LysC_rep2_E20251107-14.csv
ospf_27-239_24h_LysC_rep3_E20251107-16.csv

Each datafile is a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. Each csv file is one replicate. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_34_-_HEK293T_pervanadate-treated_chymotrypsin_phosphopeptide_library_treated_with_OspF.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with lambda phosphatase and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using chymotrypsin digestion followed by TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with OspF as described in the manuscript and quenched at 24 h
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

ospf_24h_chymotrypsin_ctrl_E20260206-06.csv - no enzyme control
ospf_24h_chymotrypsin_rep1_E20260206-08.csv
ospf_24h_chymotrypsin_rep2_E20260206-10.csv
ospf_24h_chymotrypsin_rep3_E20260206-12.csv

Each datafile is a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. Each csv file is one replicate. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_35_-_HEK293T_pervanadate-treated_chymotrypsin_phosphopeptide_library_treated_with_OspF27-239.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with lambda phosphatase and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using chymotrypsin digestion followed by TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with OspF(27-239) as described in the manuscript and quenched at 24 h
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

ospf_27-239_24h_chymotrypsin_ctrl_E20260206-06.csv - no enzyme control
ospf_27-239_24h_chymotrypsin_rep1_E20260206-14.csv
ospf_27-239_24h_chymotrypsin_rep2_E20260206-16.csv
ospf_27-239_24h_chymotrypsin_rep3_E20260206-18.csv

Each datafile is a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. Each csv file is one replicate. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_36_-_HEK293T_pervanadate-treated_trypsin_phosphopeptide_library_treated_with_PP2Ac_in_the_presence_of_okadaic_acid.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with lambda phosphatase and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using trypsin digestion followed by TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with PP2Ac in the presence of various concentrations of okadaic acid as described in the manuscript and quenched at 3 h (180 min)
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Subdirectories - replicate datasets for each timepoint are arranged into the following subdirectories:

├── dose-response/
│   ├── pp2ac_3h_OA_no_enz_ctrl_E20251111-04.csv      -no PP2Ac control
│   ├── pp2ac_3h_OA_vehicle_ctrl_E20251111-06.csv     -vehicle control
│   ├── pp2ac_3h_OA_0nM_E20251111-08.csv              -0 nM okadaic acid
│   ├── pp2ac_3h_OA_20nM_E20251111-10.csv             -20 nM okadaic acid
│   ├── pp2ac_3h_OA_30nM_E20251111-12.csv             -30 nM okadaic acid
│   ├── pp2ac_3h_OA_40nM_E20251111-14.csv             -40 nM okadaic acid
│   ├── pp2ac_3h_OA_50nM_E20251111-16.csv             -50 nM okadaic acid
│   ├── pp2ac_3h_OA_75nM_E20251111-18.csv             -75 nM okadaic acid
│   ├── pp2ac_3h_OA_100nM_E20251031-08.csv            -100 nM okadaic acid
│   ├── pp2ac_3h_OA_250nM_E20251031-10.csv            -250 nM okadaic acid
│   └── pp2ac_3h_OA_350_E20251031-12.csv              -350 nM okadaic acid
├── 20nM_OA/
│   ├── pp2ac_3h_OA_no_enz_ctrl_E20251125-06.csv      -no enzyme control
│   ├── pp2ac_3h_OA_0nM_E20251125-08.csv              -0 nM okadaic acid
│   ├── pp2ac_3h_OA_20nM_rep1_E20251125-10.csv        -20 nM okadaic acid, replicate 1
│   ├── pp2ac_3h_OA_20nM_rep2_E20251125-12.csv        -20 nM okadaic acid, replicate 2
│   └── pp2ac_3h_OA_20nM_rep3_E20251125-14.csv        -20 nM okadaic acid, replicate 3
└── 50nM_OA/
│   ├── pp2ac_3h_OA_no_enz_ctrl_E20251125-06.csv      -no enzyme control
│   ├── pp2ac_3h_OA_0nM_E20251125-08.csv              -0 nM okadaic acid
│   ├── pp2ac_3h_OA_50nM_rep1_E20251125-16.csv        -50 nM okadaic acid, replicate 1
│   ├── pp2ac_3h_OA_50nM_rep2_E20251125-18.csv        -50 nM okadaic acid, replicate 2
│   └── pp2ac_3h_OA_50nM_rep3_E20251125-20.csv        -50 nM okadaic acid, replicate 3

Each folder contains csv files corresponding to the Peptide Groups tab of search results from Proteome Discoverer. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_37_-_HEK293T_pervanadate-treated_trypsin_phosphopeptide_library_treated_with_PTP1B_in_the_presence_of_okadaic_acid.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with lambda phosphatase and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using trypsin digestion followed by TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with PTP1B in the presence of various concentrations of okadaic acid as described in the manuscript and quenched at 1 h (60 min)
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Subdirectories - replicate datasets for each timepoint are arranged into the following subdirectories:

├── 20nM_OA/
│   ├── ptp1b_60min_no_enz_ctrl_E20251120-06.csv      -no enzyme control
│   ├── ptp1b_60min_0nM_E20251120-08.csv              -0 nM okadaic acid
│   ├── ptp1b_60min_20nm_rep1_E20251120-10.csv        -20 nM okadaic acid, replicate 1
│   ├── ptp1b_60min_20nM_rep2_E20251120-12.csv        -20 nM okadaic acid, replicate 2
│   └── ptp1b_60min_20nM_rep3_E20251120-14.csv        -20 nM okadaic acid, replicate 3
└── 50nM_OA/
│   ├── ptp1b_60min_no_enz_ctrl_E20251120-06.csv      -no enzyme control
│   ├── ptp1b_60min_0nM_E20251120-08.csv              -0 nM okadaic acid
│   ├── ptp1b_60min_50nM_rep1_E20251120-16.csv        -50 nM okadaic acid, replicate 1
│   ├── ptp1b_60min_50nM_rep2_E20251120-18.csv        -50 nM okadaic acid, replicate 2
│   └── ptp1b_60min_50nM_rep3_E20251120-20.csv        -50 nM okadaic acid, replicate 3

Each folder contains csv files corresponding to the Peptide Groups tab of search results from Proteome Discoverer. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_38_-_HEK293T_pervanadate-treated_trypsin_phosphopeptide_library_treated_with_WipA_in_the_presence_of_okadaic_acid.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with lambda phosphatase and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using trypsin digestion followed by TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with WipA in the presence of various concentrations of okadaic acid as described in the manuscript and quenched at 2 h (120 min)
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Subdirectories - replicate datasets for each timepoint are arranged into the following subdirectories:

├── 20nM_OA/
│   ├── wipa_120min_no_enz_ctrl_E20251118-06.csv      -no enzyme control
│   ├── wipa_120min_0nM_E20251118-08.csv              -0 nM okadaic acid
│   ├── wipa_120min_20nM_rep1_E20251118-10.csv        -20 nM okadaic acid, replicate 1
│   ├── wipa_120min_20nM_rep2_E20251118-12.csv        -20 nM okadaic acid, replicate 2
│   └── wipa_120min_20nM_rep3_E20251118-14.csv        -20 nM okadaic acid, replicate 3
└── 50nM_OA/
│   ├── wipa_120min_no_enz_ctrl_E20251118-06.csv      -no enzyme control
│   ├── wipa_120min_0nM_E20251118-08.csv              -0 nM okadaic acid
│   ├── wipa_120min_50nM_rep1_E20251118-16.csv        -50 nM okadaic acid, replicate 1
│   ├── wipa_120min_50nM_rep2_E20251118-18.csv        -50 nM okadaic acid, replicate 2
│   └── wipa_120min_50nM_rep3_E20251118-20.csv        -50 nM okadaic acid, replicate 3

Each folder contains csv files corresponding to the Peptide Groups tab of search results from Proteome Discoverer. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_39_-_HEK293T_pervanadate-treated_trypsin_phosphopeptide_library_treated_with_WipB_in_the_presence_of_okadaic_acid.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with lambda phosphatase and analyzed at different time points.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using trypsin digestion followed by TiO2 enrichment as described in the accompanying manuscript.
Libraries were treated with WipB in the presence of various concentrations of okadaic acid as described in the manuscript and quenched at 2 h (120 min)
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Subdirectories - replicate datasets for each timepoint are arranged into the following subdirectories:

├── 20nM_OA/
│   ├── wipb_120min_no_enz_ctrl_E20251204-10.csv      -no enzyme control
│   ├── wipb_120min_0nM_E20251204-12.csv              -0 nM okadaic acid
│   ├── wipb_120min_20nM_rep1_E20251204-14.csv        -20 nM okadaic acid, replicate 1
│   ├── wipb_120min_20nM_rep2_E20251204-16.csv        -20 nM okadaic acid, replicate 2
│   └── wipb_120min_20nM_rep3_E20251204-18.csv        -20 nM okadaic acid, replicate 3
└── 50nM_OA/
│   ├── wipb_120min_no_enz_ctrl_E20251204-10.csv      -no enzyme control
│   ├── wipb_120min_0nM_E20251204-12.csv              -0 nM okadaic acid
│   ├── wipb_120min_50nM_rep1_E20251204-20.csv        -50 nM okadaic acid, replicate 1
│   ├── wipb_120min_50nM_rep2_E20251204-22.csv        -50 nM okadaic acid, replicate 2
│   └── wipb_120min_50nM_rep3_E20251204-24.csv        -50 nM okadaic acid, replicate 3

Each folder contains csv files corresponding to the Peptide Groups tab of search results from Proteome Discoverer. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_40_-_HEK293T_pervanadate-treated_phosphopeptide_library_Fe-NTA_enrichment.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for 4 replicate phosphoproteome-derived peptide libraries made from pervanadate-treated HEK293T cells.

Data collection and analysis:

Phosphoproteome-derived peptide libraries were prepared using tryptic digestion followed by Fe-NTA enrichment as described in the accompanying manuscript.
LC-MS/MS data were collected on an Orbitrap Exploris 480 (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Site localization: Phosphosite localization was scored using ptmRS
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Datafiles:

HEK293T_pervanadate_Fe-NTA_rep1_E20260114-06.csv
HEK293T_pervanadate_Fe-NTA_rep2_E20260114-08.csv
HEK293T_pervanadate_Fe-NTA_rep3_E20260114-10.csv
HEK293T_pervanadate_Fe-NTA_rep4_E20260114-12.csv

Each file contains a csv file corresponding to the Peptide Groups tab of search results from Proteome Discoverer. The columns in each csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
# Missed Cleavages
Theo. MH+ [Da]
Found in Sample: [S10] F10: Sample
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT

File: Supplementary_Dataset_41_-_OspF_induced_vs_uninduced_LFQ_results.zip

Description:

This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with OspF variants.

Data collection and analysis:

Phospho-enriched LFQ samples were prepared as described in the accompanying manuscript.
LC-MS/MS data were collected on an Orbitrap Exploris 480 mass spectrometer (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Files:

E20251008-OspF_LFQ_results.csv --Peptide Groups tab of search results from Proteome Discoverer
E20251008-OspF_LFQ_summary_results.csv --Summary results:

Columns in each file:

E20251008-OspF_LFQ_results.csv corresponds to the Peptide Groups tab of search results from Proteome Discoverer. The columns in the csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
Master Protein Descriptions
# Missed Cleavages
Theo. MH+ [Da]
log2 Abundance Ratio: (induced) / (non-induced)
Abundance Ratio: (induced) / (non-induced)
neg log Abundance Ratio P-Value: (induced) / (non-induced)
Abundance Ratio P-Value: (induced) / (non-induced)
Abundance Ratio Adj. P-Value: (induced) / (non-induced)
Abundances (Grouped): induced
Abundances (Grouped): non-induced
Abundances (Grouped) CV [%]: induced
Abundances (Grouped) CV [%]: non-induced
Abundances (Scaled): F2: Sample, induced
Abundances (Scaled): F4: Sample, induced
Abundances (Scaled): F6: Sample, induced
Abundances (Scaled): F8: Sample, induced
Abundances (Scaled): F1: Sample, non-induced
Abundances (Scaled): F3: Sample, non-induced
Abundances (Scaled): F5: Sample, non-induced
Abundances (Scaled): F7: Sample, non-induced
Abundances (Normalized): F2: Sample, induced
Abundances (Normalized): F4: Sample, induced
Abundances (Normalized): F6: Sample, induced
Abundances (Normalized): F8: Sample, induced
Abundances (Normalized): F1: Sample, non-induced
Abundances (Normalized): F3: Sample, non-induced
Abundances (Normalized): F5: Sample, non-induced
Abundances (Normalized): F7: Sample, non-induced
Abundance: F2: Sample, induced
Abundance: F4: Sample, induced
Abundance: F6: Sample, induced
Abundance: F8: Sample, induced
Abundance: F1: Sample, non-induced
Abundance: F3: Sample, non-induced
Abundance: F5: Sample, non-induced
Abundance: F7: Sample, non-induced
Quan Info
Found in Sample: [S2] F2: Sample, induced
Found in Sample: [S4] F4: Sample, induced
Found in Sample: [S6] F6: Sample, induced
Found in Sample: [S8] F8: Sample, induced
Found in Sample: [S1] F1: Sample, non-induced
Found in Sample: [S3] F3: Sample, non-induced
Found in Sample: [S5] F5: Sample, non-induced
Found in Sample: [S7] F7: Sample, non-induced
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT
Top Apex RT [min]

E20251008-OspF_LFQ_summary_results.csv is a summary of the results across four induced and four uninduced replicates. The columns in this file are:

Modifications in Master Proteins - Describes the position of the peptide and modified sites in the protein to which the peptide was assigned. For example, P16949 2xPhospho [S31; S38] describes the protein with the Uniprot ID P16949 that is phosphorylate on Ser 31 and Ser 38
Master Protein Descriptions - Describes which protein the peptide comes from. See additional information below
log2 Abundance Ratio: (induced) / (non-induced) - Mean abundance ratio for OspF induced vs uninduced across the replicates
-log10 P-Value: (induced) / (non-induced) - Abundance ratio P-Value

Additional information about the Master Protein Descriptions column: This column contains a description of the protein, and additional annotations using SwissProt-style headers as follows

OS = Organism Species

→ The species from which the protein originates

OS = Homo sapiens
OX = Organism taxonomy ID

→ NCBI taxonomy identifier

OX = 9606 (9606 = human)
GN = Gene Name
GN = TTK
PE = Protein Existence

→ Level of evidence supporting the existence of the protein

PE = 1 means evidence at the protein level (highest confidence)

UniProt PE levels:

1 — Evidence at the protein level
2 — Evidence at the transcript level
3 — Inferred from homology
4 — Predicted
5 — Uncertain

SV = Sequence Version

→ Version number of the protein sequence

SV = 2 means the sequence has been updated once since initial entry

File: Supplementary_Dataset_42_-_OspF_induced_vs_uninduced_in_EGF_treated_cells_LFQ_results.zip

Description: This dataset accompanies the manuscript "Phosphoproteome-derived peptide libraries for deep specificity profiling of enzymatic phosphorylation erasers". This dataset contains csv files containing peptide identification search results for replicate phosphoproteome-derived peptide libraries made from HEK293T cells that were treated with OspF variants.

Data collection and analysis:

Phospho-enriched LFQ samples were prepared as described in the accompanying manuscript.
LC-MS/MS data were collected on an Orbitrap Lumos mass spectrometer (ThermoFisher Scientific)
Search: Database search was performed with Proteome Discoverer v2.4 using SEQUEST HT with Percolator post-processing
Database: SwissProt human (downloaded January 24, 2020)

How the data are organized:

README.txt — This file

Files:

20260108_OspF_EGFtreatment_LFQ_peptideGroups.csv - Peptide Groups tab of search results from Proteome Discoverer
20260108_OspF_EGFtreatment_LFQ_summary_results.csv - Summary results

Columns in each file:

20260108_OspF_EGFtreatment_LFQ_peptideGroups.csv corresponds to the Peptide Groups tab of search results from Proteome Discoverer. The columns in the csv are:

Checked
Confidence
Annotated Sequence - this column is used in downstream analysis to extract the peptide sequence
Modifications - this column contains information about the localization of phosphorylation, dehydration, and other modifications. An example is '1xPhospho [S17(98.6)]', which describes a peptide with one phosphosite at positions S17 with a ptmRS site localization score of 98.6
Qvality PEP
Qvality q-value
# Protein Groups
# Proteins
# PSMs
Master Protein Accessions - Uniprot accession numbers
Positions in Master Proteins
Modifications in Master Proteins - this column contains information about the location of modifications within the protein from which the peptide is derived. An example is 'P05387 1xPhospho [S17(98.6)]', where P05387 is the protein that this peptide is derived from, the peptide has one phosphorylation site at position S17 in the protein sequence, and the ptmRS site localization score is 98.6
Master Protein Descriptions
# Missed Cleavages
Theo. MH+ [Da]
log2 Abundance Ratio: (induced) / (non-induced)
Abundance Ratio: (induced) / (non-induced)
neg log Abundance Ratio P-Value: (induced) / (non-induced)
Abundance Ratio P-Value: (induced) / (non-induced)
Abundance Ratio Adj. P-Value: (induced) / (non-induced)
Abundances (Grouped): induced
Abundances (Grouped): non-induced
Abundances (Grouped) CV [%]: induced
Abundances (Grouped) CV [%]: non-induced
Abundances (Scaled): F2: Sample, induced
Abundances (Scaled): F4: Sample, induced
Abundances (Scaled): F6: Sample, induced
Abundances (Scaled): F8: Sample, induced
Abundances (Scaled): F1: Sample, non-induced
Abundances (Scaled): F3: Sample, non-induced
Abundances (Scaled): F5: Sample, non-induced
Abundances (Scaled): F7: Sample, non-induced
Abundances (Normalized): F2: Sample, induced
Abundances (Normalized): F4: Sample, induced
Abundances (Normalized): F6: Sample, induced
Abundances (Normalized): F8: Sample, induced
Abundances (Normalized): F1: Sample, non-induced
Abundances (Normalized): F3: Sample, non-induced
Abundances (Normalized): F5: Sample, non-induced
Abundances (Normalized): F7: Sample, non-induced
Abundance: F2: Sample, induced
Abundance: F4: Sample, induced
Abundance: F6: Sample, induced
Abundance: F8: Sample, induced
Abundance: F1: Sample, non-induced
Abundance: F3: Sample, non-induced
Abundance: F5: Sample, non-induced
Abundance: F7: Sample, non-induced
Quan Info
Found in Sample: [S2] F2: Sample, induced
Found in Sample: [S4] F4: Sample, induced
Found in Sample: [S6] F6: Sample, induced
Found in Sample: [S8] F8: Sample, induced
Found in Sample: [S1] F1: Sample, non-induced
Found in Sample: [S3] F3: Sample, non-induced
Found in Sample: [S5] F5: Sample, non-induced
Found in Sample: [S7] F7: Sample, non-induced
Confidence (by Search Engine): Sequest HT
Percolator q-Value (by Search Engine): Sequest HT
Percolator PEP (by Search Engine): Sequest HT
XCorr (by Search Engine): Sequest HT
Top Apex RT [min]

20260108_OspF_EGFtreatment_LFQ_summary_results.csv is a summary of the results across four induced and four uninduced replicates. The columns in this file are:

Modifications in Master Proteins - Describes the position of the peptide and modified sites in the protein to which the peptide was assigned. For example, P16949 2xPhospho [S31; S38] describes the protein with the Uniprot ID P16949 that is phosphorylate on Ser 31 and Ser 38
Master Protein Descriptions - Describes which protein the peptide comes from. See additional information below
log2 Abundance Ratio: (induced) / (non-induced) - Mean abundance ratio for OspF induced vs uninduced across the replicates
-log10 P-Value: (induced) / (non-induced) - Abundance ratio P-Value

Additional information about the Master Protein Descriptions column: This column contains a description of the protein, and additional annotations using SwissProt-style headers as follows

OS = Organism Species

→ The species from which the protein originates

OS = Homo sapiens
OX = Organism taxonomy ID

→ NCBI taxonomy identifier

OX = 9606 (9606 = human)
GN = Gene Name
GN = TTK
PE = Protein Existence

→ Level of evidence supporting the existence of the protein

PE = 1 means evidence at protein level (highest confidence)

UniProt PE levels:

1 — Evidence at protein level
2 — Evidence at transcript level
3 — Inferred from homology
4 — Predicted
5 — Uncertain

SV = Sequence Version

→ Version number of the protein sequence

SV = 2 means the sequence has been updated once since initial entry

Code/software

Alphafold3 models can be opened in protein structure viewing software such as PyMol or ChimeraX. *.pse files can be opened with PyMol.
Plasmids maps in GenBank format can be imported into Benchling, an electronic lab notebook program, and analyzed. They are also compatible with many other software for editing plasmid maps (e.g., ApE, SnapGene).
Fluorescence microscopy images (*.nd2 files) can be opened with the free software Fiji.
Uncropped Western blot images (*.tif files) can be opened with the free software Fiji.

Access information

Other publicly accessible locations of the data:

Data was derived from the following sources:

Data from: Phosphoproteome-derived peptide libraries for deep specificity profiling of phosphatases and phospholyases

Data files

Abstract

README: Data from: Phosphoproteome-derived peptide libraries for deep specificity profiling of phosphatases and phospholyases

Description of the data and file structure

Files and variables

File: Plasmid_maps.zip

File: Fluorescence_microscopy_data.zip

File: Uncropped_Western_blots.zip

File: Supplementary_Dataset_1_-_HEK293T_untreated_phosphopeptide_library.zip

File: Supplementary_Dataset_2_-_HEK293T_pervanadate-treated_phosphopeptide_library.zip

File: Supplementary_Dataset_2_-_HEK293T_pervanadate-treated_phosphopeptide_library.zip

File: Supplementary_Dataset_3_-_Jurkat_pervanadate-treated_phosphopeptide_library.zip

File: Supplementary_Dataset_4_-_K562_pervanadate-treated_phosphopeptide_library.zip

File: Supplementary_Dataset_5_-_Average_kinase_substrate_counts.zip

File: Supplementary_Dataset_6_-_HEK293T_pervanadate-treated_phosphopeptide_library_treated_with_lambda_phosphatase.zip

File: Supplementary_Dataset_7_-_HEK293T_pervandate-treated_phosphopeptide_library_treated_with_PTP1B_catalytic_domain.zip

File: Supplementary_Dataset_8_-_HEK293T_pervanadate-treated_phosphopeptide_library_treated_with_PP2Ac.zip

File: Supplementary_Dataset_9_-_HEK293T_pervanadate-treated_library_treated_with_WipA.zip

File: Supplementary_Dataset_10_-_HEK293T_pervanadate-treated_library_treated_with_WipB.zip

File: Supplementary_Dataset_11_-_HEK293T_pervanadate-treated_library_treated_with_OspF.zip

File: Supplementary_Dataset_12_-_HEK293T_pervanadate-treated_library_treated_with_SpvC_or_HopAI.zip

File: Supplementary_Dataset_13_-_HEK293T_pervanadate-treated_library_treated_with_OspF_27-239.zip

File: Supplementary_Dataset_14_-_OspF_TMT_results.zip

File: Supplementary_Dataset_15_-_OspF_alanine_variant_activity_on_HEK293T_phosphopeptide_library.zip

File: Supplementary_Dataset_16_-_Additional_OspF_variant_activity_on_HEK293T_phosphopeptide_library.zip

File: Supplementary_Dataset_17_-_HEK293T_pervanadate-treated_phosphopeptide_library_GluC.zip

File: Supplementary_Dataset_18_-_HEK293T_pervanadate-treated_phosphopeptide_library_LysC.zip

File: Supplementary_Dataset_19_-_HEK293T_pervanadate-treated_phosphopeptide_library_chymotrypsin.zip

File: Supplementary_Dataset_20_-_HEK293T_pervanadate-treated_GluC_phosphopeptide_library_treated_with_lambda_phosphatase.zip

File: Supplementary_Dataset_21_-_HEK293T_pervanadate-treated_GluC_phosphopeptide_library_treated_with_PP2Ac.zip

File: Supplementary_Dataset_22_-_HEK293T_pervanadate-treated_GluC_phosphopeptide_library_treated_with_PTP1B.zip

File: Supplementary_Dataset_23_-_HEK293T_pervanadate-treated_GluC_phosphopeptide_library_treated_with_WipA.zip

File: Supplementary_Dataset_24_-_HEK293T_pervanadate-treated_GluC_phosphopeptide_library_treated_with_WipB.zip

File: Supplementary_Dataset_25_-_HEK293T_pervanadate-treated_GluC_phosphopeptide_library_treated_with_OspF.zip

File: Supplementary_Dataset_26_-_HEK293T_pervanadate-treated_GluC_phosphopeptide_library_treated_with_OspF27-239.zip

File: Supplementary_Dataset_27_-_HEK293T_pervanadate-treated_LysC_phosphopeptide_library_treated_with_lambda_phosphatase.zip

File: Supplementary_Dataset_28_-_HEK293T_pervanadate-treated_LysC_phosphopeptide_library_treated_with_PP2Ac.zip

File: Supplementary_Dataset_29_-_HEK293T_pervanadate-treated_LysC_phosphopeptide_library_treated_with_PTP1B.zip

File: Supplementary_Dataset_30_-_HEK293T_pervanadate-treated_LysC_phosphopeptide_library_treated_with_WipA.zip

File: Supplementary_Dataset_31_-_HEK293T_pervanadate-treated_LysC_phosphopeptide_library_treated_with_WipB.zip

File: Supplementary_Dataset_32_-_HEK293T_pervanadate-treated_LysC_phosphopeptide_library_treated_with_OspF.zip

File: Supplementary_Dataset_33_-_HEK293T_pervanadate-treated_LysC_phosphopeptide_library_treated_with_OspF27-239.zip

File: Supplementary_Dataset_34_-_HEK293T_pervanadate-treated_chymotrypsin_phosphopeptide_library_treated_with_OspF.zip

File: Supplementary_Dataset_35_-_HEK293T_pervanadate-treated_chymotrypsin_phosphopeptide_library_treated_with_OspF27-239.zip

File: Supplementary_Dataset_36_-_HEK293T_pervanadate-treated_trypsin_phosphopeptide_library_treated_with_PP2Ac_in_the_presence_of_okadaic_acid.zip

File: Supplementary_Dataset_37_-_HEK293T_pervanadate-treated_trypsin_phosphopeptide_library_treated_with_PTP1B_in_the_presence_of_okadaic_acid.zip

File: Supplementary_Dataset_38_-_HEK293T_pervanadate-treated_trypsin_phosphopeptide_library_treated_with_WipA_in_the_presence_of_okadaic_acid.zip

File: Supplementary_Dataset_39_-_HEK293T_pervanadate-treated_trypsin_phosphopeptide_library_treated_with_WipB_in_the_presence_of_okadaic_acid.zip

File: Supplementary_Dataset_40_-_HEK293T_pervanadate-treated_phosphopeptide_library_Fe-NTA_enrichment.zip

File: Supplementary_Dataset_41_-_OspF_induced_vs_uninduced_LFQ_results.zip

File: Supplementary_Dataset_42_-_OspF_induced_vs_uninduced_in_EGF_treated_cells_LFQ_results.zip

Code/software

Access information