Dataset DOI: 10.5061/dryad.j3tx95xwc

Description of the data and file structure

DisciplineSpecificMetadata.json contains information about the Mass Spectrometry experimental methodology including: 

• Preparation protocol DOI
• Dataset type
• Analyte class
• Targeted approach
• Acquisition instrument vendor
• MS ionization technique
• MS scan mode
• Mass analysis polarity

Phosphoproteomics data is contained within csv files.

Data from experiments using Chymotrypsin cleavage during sample preparation can be found within the following files: 

• Lea-Case_Chymotrypsin_Report-AllPeptideSummary.csv
• Lea-Case_Chymotrypsin_Report-PhosphoPeptideSummary.csv
• Lea-Case_Chymotrypsin_Report-Proteins.csv
• Lea-Case_Chymotrypsin_Report-PSMs.csv
• Lea-Case_Chymotrypsin_Report-sort.csv
• Lea-Case_Chymotrypsin_Report-sumpep.csv

Data from experiments using Trypsin cleavage during sample preparation can be found within the following files: 

• Lea-Case_Trypsin_Report-AllPeptidesSummary.csv
• Lea-Case_Trypsin_Report-PhosphoPepSummary.csv
• Lea-Case_Trypsin_Report-Proteins.csv
• Lea-Case_Trypsin_Report-PSMs.csv
• Lea-Case_Trypsin_Report-sort.csv
• Lea-Case_Trypsin_Report-SumPept.csv

Variables and abbreviations in the files:

Sample Name: description of the experimental conditions for each detected peptide

Protein Accessions: Accession number for each detected peptide. Uniprot accession numbers.

Protein Descriptions: Descriptions of proteins for each detected peptide

Sequence: sequence of the peptide

Modifications: Post-translational modifications (PTMs) detected on the peptide, including modification type (Phospho, Oxidation,or Carbamidomethyl), amino acid (using single amino acid code) and position. (Abbreviations used: Phospho = Phosphorylation, Oxidation = Oxidation, Carbamidomethyl = Carbamidomethylation). Blank values indicate no detected postranslational modification.

Sum of Intensity: Quantitative measure of peptide/protein abundance, calculated as the sum of precursor ion intensities across all spectra. Higher values indicate greater abundance.

Total Spectra: Number of MS/MS spectra (peptide spectrum matches) assigned to this protein/peptide. Higher counts indicate more confident identifications and more abundant species.

Accession: UniProt accession number for each protein detected in the sample

Description: Full name and functional description of the protein, including gene name and organism. Provides context for protein identity.

MW [kDa]: Molecular weight of the protein in kilodaltons (kDa).

Score Sequest HT: Sequest HT database search engine match score for the protein in each sample. Higher scores indicate greater confidence in protein identification. Blank value indicates the protein was not detected in the indicated sample. 

# PSMs (by Search Engine): "Number of MS/MS spectra matched to peptides from this protein by Sequest HT in each experimental sample (PSM = Peptide Spectrum Match). Higher PSM counts indicate greater protein abundance and detection confidence. Blank value indicates the protein was not detected in the indicated sample. 

# Peptides (by Search Engine): Number of unique/distinct peptide sequences identified for this protein in each experimental sample. Indicates protein sequence coverage and detection diversity. Blank value indicates the protein was not detected in the indicated sample.

Checked: Manual validation flag (True/False). Indicates whether this PSM has been manually reviewed and confirmed by the analyst.

Confidence: Classification of identification confidence level (High, Medium, or Low). Based on search engine scores and statistical thresholds.

Identifying node: The search engine used to identify the peptide.

PSM Ambiguity: Indicates whether this MS/MS spectrum uniquely matches one peptide sequence or could match multiple sequences. Unambiguous indicates a unique match. 

Sequence: Amino acid sequence of the identified peptide.

Annotated Sequence: Amino acid sequence with modification symbols annotated. Lower case letter indicates where modifications occur on the peptide.

Modifications: Detailed list of all post-translational modifications detected on this peptide, including position and type (e.g., Phospho (S4); Oxidation (M1)). Blank value means there was no modification detected (no PTM on the peptide)

# Proteins: Number of proteins this peptide sequence is mapped to. Used to assess peptide specificity.

Master Protein Accessions: The primary/canonical protein accession assigned to this peptide.

Protein Accessions: All protein database accession numbers to which this peptide maps.

Protein Descriptions: Full names and functional descriptions of all proteins identified, including gene names and organisms.

# Missed Cleavages: Number of trypsin or chymotrypsin cleavage sites on this peptide that were NOT cleaved during sample preparation.

Charge: Charge state (+1, +2, +3, etc.) of the peptide ion detected by the mass spectrometer.

DeltaScore: Score difference between the top-scoring match and the second-best match for this spectrum.

DeltaCn: Normalized correlation difference (Sequest-specific metric).

Rank: Overall ranking of this PSM among all PSMs in the analysis. Rank 1 = the best-scoring PSM in the entire dataset.

Search Engine Rank: Ranking of this PSM within the specific search engine results (as opposed to overall ranking).

m/z [Da]: Observed mass-to-charge ratio of the peptide ion in Daltons (Da).

MH+ [Da]: Observed monoisotopic mass of the peptide plus one proton (M+H), calculated from the observed m/z and charge state. The "true" mass of the detected ion.

Theo. MH+ [Da]: Theoretical monoisotopic mass of the peptide (based on amino acid composition) plus one proton. What the mass "should be" for the identified sequence.

DeltaM [ppm]: Mass error expressed in parts per million (ppm). Calculated as (Observed mass – Theoretical mass) / Theoretical mass × 10⁶.

Deltam/z [Da]: Mass error expressed in Daltons (Da). Calculated as Observed m/z – Theoretical m/z.

Intensity: Peak intensity/abundance of the detected precursor ion.

Activation Type: Ionization/fragmentation method used. HCD = Higher-energy Collision Dissociation.

NCE [%]: Normalized Collision Energy (percentage). The energy used to fragment the peptide ion during MS/MS.

MS Order: MS level of the scan. MS2 is MS/MS, fragmentation spectra used for peptide identification.

Isolation Interference [%]: Percentage of unwanted ions (contaminating m/z values) in the selected precursor ion isolation window. Lower is better. 

Ion Inject Time [ms]: Time (in milliseconds) that the mass spectrometer allowed ions to accumulate in the ion trap before analysis.

RT [min]: Retention time in minutes. When (during the liquid chromatography gradient) this peptide eluted from the column.

First Scan: Scan number of the first/primary MS/MS spectrum for this PSM.

Spectrum File: Name of the raw data file (.raw or .mzML) from which this spectrum originated.

File ID: Unique identifier for the spectrum file.

XCorr: Cross-correlation score from Sequest HT database search engine

# Protein Groups: Number of protein groups to which this peptide belongs. A "protein group" is a set of proteins that cannot be distinguished based on the peptide evidence (due to sequence homology or shared peptides).

Percolator q-Value: False Discovery Rate (FDR)-adjusted q-value computed by Percolator statistical validation algorithm. Represents the probability that this PSM is a false positive.

Percolator PEP: Posterior Error Probability from Percolator. Probability that this specific PSM is incorrect, given all available evidence.

Spectrum File: Experimental condition the peptide originated from.

Protein Accessions: UniProt accession number(s) for the protein(s) to which this peptide maps.

Protein Descriptions: Full name and functional description of the identified protein(s), including gene name and organism.

Sequence: Amino acid sequence of the identified peptide.

Modifications: Post-translational modifications (PTMs) detected on this peptide, including modification type, amino acid residue, and position.

Intensity: Peak intensity (signal strength) of this peptide's precursor ion. Higher values indicate greater peptide abundance in the sample.

Tot Pep: How many times this peptide was detected in the sample.

Sum of Intensity: Total/summed intensity from all MS/MS spectra detected for this peptide.

Sum of Tot Pep: Total count of all times this peptide was detected across all spectra.

Experimental Conditions of the Analyzed Samples: 

There are 6 experimental conditions in each experiment:

1. Abl dephosphorylated - Recombinant Abl dephosphorylated in vitro and purified with gel filtration.

2. Abl phosphorylated - Recombinant Abl that was autophosphorylated inside Abl condensates.

3. FAK dephosphorylated - Recombinant FAK dephosphorylated in vitro and purified with gel filtration.

4. FAK phosphorylated - Recombinant FAK that was autophosphorylated inside FAK condensates

5. MST2 dephosphorylated - Recombinant MST2 dephosphorylated in vitro and purified with gel filtration.

6. MST2 phosphorylated - Recombinant MST2 that was autophosphorylated inside condensates containing MST2 and SAV1.

Each dataset contains all the phospho peptides identified in each of the sample as well as phospho peptides detected after FeNTA enrichment (annotated as FeElution). For more experimental details, please refer to the associated paper.

For the data in our paper, we analyzed the Phospho Peptide Summary, which contains the final analyzed data for each experiment, and can be found in the files: Lea-Case_Chymotrypsin_Report-PhosphoPeptideSummary.csv and Lea-Case_Trypsin_Report-PhosphoPepSummary.csv.

Accession Numbers:

In addition to UniProt accession numbers, the recombinant protein sequences we used in this study were added to a Human database using the following Protein Accessions:

Abl protein=ABLNL

FAK protein= FAKNL

MST2 protein= MST2NL

SAV1 protein= SAV1NL

Missing or Empty Values

Some columns in the dataset contain empty cells. These are retained in their original format and are not errors or omissions. Instead, they reflect values that were not identified in the experiment. Below is a summary of common cases:

Modifications: an empty cell reflects a peptide with no post translational modification detected

Score Sequest HT: an empty cell indicates the protein was not detected in the indicated sample. 

# PSMs (by Search Engine): an empty cell indicates the protein was not detected in the indicated sample. 

# Peptides (by Search Engine): an empty cell indicates the protein was not detected in the indicated sample.