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Skillstooluniverse-protein-modification-analysis

tooluniverse-protein-modification-analysis

Data & analytics

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Updated 10-15 17:06

About this skill

Protein Post-Translational Modification Analysis

Comprehensive PTM analysis using iPTMnet (primary), ProtVar (functional context), UniProt (baseline), STRING (interactions), ELM (linear motifs), and MassIVE/ProteomeXchange (experimental data).

iPTMnet is down (checked 2026-10-08). Its REST API has answered HTTP 503 to every request since at least 2026-09-20 while the website stays up, so every iPTMnet_* call below currently returns that error. Try one call; if it is a 503, use the fallbacks given in each phase -- UniProt_get_ptm_processing_by_accession for modified residues (with the modifying enzyme where curated), OmniPath_get_enzyme_substrate for enzyme-substrate pairs, EBIProteins_get_proteomics_ptm for mass-spectrometry evidence. Nothing replaces iPTMnet's proteoform and PTM-dependent-interaction data; say so in the report rather than filling the gap.

LOOK UP DON'T GUESS

  • PTM sites/enzymes: iPTMnet_get_ptm_sites
  • Functional consequence: ProtVar_get_function + iPTMnet_get_ptm_ppi
  • Proteoforms: iPTMnet_get_proteoforms
  • Linear motifs: ELM_get_instances

COMPUTE, DON'T DESCRIBE

When analysis requires computation (statistics, data processing, scoring, enrichment), write and run Python code via Bash. Don't describe what you would do — execute it and report actual results. Use ToolUniverse tools to retrieve data, then Python (pandas, scipy, statsmodels, matplotlib) to analyze it.

Domain Reasoning

PTMs are context-dependent: same phosphorylation site can activate or inhibit depending on kinase and effectors. Always check: which enzyme, what functional consequence, in what cell context.

KEY PRINCIPLES

  1. Disambiguation first -- resolve to UniProt accession before iPTMnet calls
  2. iPTMnet is SOAP-style -- every call requires operation parameter
  3. Evidence-graded -- distinguish experimental (T1) from predicted (T4)
  4. English-first queries

Workflow

Phase 0: Protein Disambiguation → UniProt accession
Phase 1: PTM Site Inventory → iPTMnet_get_ptm_sites
Phase 2: Proteoform Analysis → iPTMnet_get_proteoforms
Phase 3: PTM-Dependent Interactions → iPTMnet_get_ptm_ppi
Phase 4: Functional Context → ProtVar_get_function at key sites
Phase 4b: Linear Motif Context → ELM_get_instances for SLiM overlap
Phase 4c: Experimental Data → MassIVE/ProteomeXchange
Phase 5: Synthesis & Report

Phase 0: Disambiguation

  • iPTMnet_search(operation="search", search_term="TP53", role="Substrate") -- find UniProt IDs
  • If user provides UniProt accession directly, use it
  • If iPTMnet is down: UniProt_search(query="gene_exact:TP53 AND reviewed:true", organism="human", limit=3) for the accession (a bare "TP53" ranks TP53TG5 and TP53BP1 above P04637)
  • Select human entry if multiple hits

Phase 1: PTM Sites

iPTMnet_get_ptm_sites(operation="get_ptm_sites", uniprot_id="P04637") -- returns position, residue, modification type, enzyme, evidence. Group by modification type. Fallback: UniProt_get_ptm_processing_by_accession(accession="P04637") (modified residues, with the modifying enzyme in the description where curated, e.g. "Phosphoserine; by HIPK4"), OmniPath_get_enzyme_substrate(substrates="TP53") for enzyme-substrate pairs with residue and modification type, and EBIProteins_get_proteomics_ptm(accession="P04637") for MS-observed sites.

Phase 2: Proteoforms

iPTMnet_get_proteoforms(operation="get_proteoforms", uniprot_id=...) -- distinct PTM combinations. Focus on those with functional/disease annotations if >20.

Phase 3: PTM-Dependent Interactions

iPTMnet_get_ptm_ppi(operation="get_ptm_ppi", uniprot_id=...) -- interacting protein, PTM site, effect (enables/disrupts). Supplement with STRING_get_interaction_partners(identifiers=gene, species=9606, required_score=700). If iPTMnet is down, OmniPath_get_enzyme_substrate(enzymes=gene) and (substrates=gene) give the enzyme-substrate pairs behind many PTM-dependent interactions, but not whether a modification enables or disrupts a binding.

Phase 4: Functional Context

ProtVar_get_function(accession=..., position=N, variant_aa=AA) -- domain, active site, binding site, conservation. Grade: active-site PTM > domain-core > disordered region.

Phase 4b: Linear Motifs (ELM)

ELM_get_instances(operation="get_instances", uniprot_id=..., motif_type="MOD") -- MOD = modification sites, DEG = degradation signals. Cross-reference with Phase 1 PTM positions. ELM_list_classes(operation="list_classes") for motif details.

Phase 4c: Experimental Data

MassIVE_search_datasets(species="9606"), MassIVE_get_dataset(accession="MSV...") for public MS datasets.

Phase 4d: EBI Proteins Cross-Validation (Structural, Antigenic, MS-Peptide Evidence)

src/tooluniverse/data/ebi_proteins_ext_tools.json provides 11 UniProt-accession-keyed tools from the EBI Proteins API — a second, independent evidence source to corroborate or extend the iPTMnet/ProtVar findings above, not a replacement for them:

Tool Returns When to use
EBIProteins_get_mutagenesis Experimentally characterized point-mutation effects (gain/loss of function, binding/structural impact), UniProtKB-curated with literature evidence Corroborate a PTM-adjacent residue's functional importance with direct mutagenesis evidence, independent of iPTMnet
EBIProteins_get_proteomics_ptm MS-evidence-backed PTM sites from PeptideAtlas/ProteomicsDB/MaxQB/EPD Cross-check an iPTMnet PTM call against raw MS detection evidence — this is detection evidence, not curated/annotated PTM biology, so treat a hit here as "observed by MS" not "known to be functional"
EBIProteins_get_features Consolidated UniProt features by category (DOMAINS_AND_SITES, MOLECULE_PROCESSING, PTM, STRUCTURAL, TOPOLOGY, VARIANTS, MUTAGENESIS; default DOMAINS_AND_SITES) Get all UniProt-curated PTM annotations in one call via category="PTM" — a faster alternative to browsing full UniProt when only PTM feature rows are needed
EBIProteins_get_antigen Predicted antigenic regions with match-confidence scores PTM sites near/inside an antigenic region can affect antibody binding — relevant when a PTM study feeds into antibody or immunoassay design
EBIProteins_get_coordinates UniProt protein -> Ensembl gene/transcript ID, chromosome, strand, exon count Map a PTM residue position back to genomic coordinates for genome-browser cross-referencing
EBIProteins_get_proteomics_peptides Generic MS peptide-level detection evidence (position, uniqueness, source DB) Confirm a PTM-bearing region of the protein has actually been observed by mass spec at all, before trusting a PTM call there
EBIProteins_get_hpp_peptides Human Proteome Project peptide evidence — a stringent, typically larger evidence set than get_proteomics_peptides for the same accession Prefer this over get_proteomics_peptides when the strongest available MS confirmation is needed (human proteins only)

Known broken (verified live, not a guess): EBIProteins_get_rna_editing returns HTTP 404 directly from https://www.ebi.ac.uk/proteins/api/rna_editing/... for both of its own documented example accessions (P42262, P28335), while the sibling mutagenesis endpoint on the same API returns 200 for the same host. This looks like EBI deprecated/removed the endpoint rather than a transient outage — re-verify with a direct tu run EBIProteins_get_rna_editing '{"accession": "..."}' before relying on it, and do not present RNA-editing results from this tool as available without that check.

Protein-level variant lookup (distinct from Phase 1-4 PTM tools, not duplicative of tooluniverse-variant-analysis's SPDI/HGVS/rsID notation conversion):

Tool Returns When to use
EBIProteins_get_variation All known variants for a UniProt accession, merged from COSMIC/ClinVar/gnomAD/ExAC/UniProt, with clinical significance and disease associations; filterable by source_type and disease_only Want every reported variant on a protein with disease context in one call
EBIProteins_get_variation_by_dbsnp Reverse lookup: given a dbSNP rsID, every UniProt entry (incl. isoforms) carrying that variant, with the mapped protein-level consequence Have an rsID, need to know which protein(s)/isoform(s) it affects and how — tooluniverse-variant-analysis's NCBIVariation_* tools convert rsID<->SPDI<->HGVS notation but do not resolve to a UniProt protein consequence the way this does
EBIProteins_get_variation_by_hgvs Reverse lookup: given an HGVS genomic expression (NC_...:g....), every overlapping UniProt entry with the resulting protein-level consequence Have a genomic HGVS change, need its amino-acid consequence across all affected isoforms in one call, rather than converting notation first and then looking up each protein separately

All 3 return multiple UniProt entries per query (isoforms, paralogs sharing the position) — always check total_entries/total_variants and report which specific entry/isoform a finding came from, don't assume the first entry is the canonical one.

Single-category shortcuts (verified identical data to EBIProteins_get_features): EBIProteins_get_domains_sites, EBIProteins_get_molecule_processing, and EBIProteins_get_structural_features each take a plain accession and return exactly the same feature list as EBIProteins_get_features(accession, category="DOMAINS_AND_SITES"|"MOLECULE_PROCESSING"|"STRUCTURAL") respectively — confirmed live on P04637, byte-for-byte the same 31 DOMAINS_AND_SITES features (minor field-naming difference only: the dedicated tool flattens evidences[].source/.id, the generic one nests source_name/source_id). Use the dedicated tool when you already know which single category you want (one fewer parameter); use EBIProteins_get_features when browsing multiple categories in a loop. EBIProteins_get_molecule_processing is the one to reach for on precursor proteins that get cleaved into a mature form — e.g. P01308 (insulin) returns signal peptide (1-24), B chain (25-54), C peptide (57-87), A chain (90-110).

Protein-to-genome coordinate mapping (two tools, more detail than the existing EBIProteins_get_coordinates summary above):

Tool Direction Returns
EBIProteins_get_coordinate_mapping protein → genome Per-exon residue-to-genomic-coordinate mapping across all transcript isoforms (chromosome, strand, exon boundaries) — exon-level detail that EBIProteins_get_coordinates (chromosome/strand/exon count only) doesn't give you
EBIProteins_get_proteins_by_genomic_loc genome → protein Reverse lookup: given taxonomy + location ("17:7676154", 1-based) or chromosome+position, every UniProt protein whose coding sequence overlaps that position, with the exact affected residue

Real example (verified live): EBIProteins_get_coordinate_mapping(accession="P04637") → TP53 maps to 6 transcripts on chromosome 17 (reverse strand), 10 exons, positions 7669612-7676594. The reverse direction confirms it: EBIProteins_get_proteins_by_genomic_loc(taxonomy="9606", location="17:7676154") → TP53 (P04637) at protein residue 72 (Pro) on transcript ENST00000923569 — use this when you have a chromosomal variant position and need to know which protein(s)/residue it hits, before running any PTM/functional analysis on that residue.

Epitopes (experimental, IEDB-sourced — distinct from EBIProteins_get_antigen's predicted antigenic regions): EBIProteins_get_epitopes(accession) returns experimentally-mapped immune epitope regions with sequence, PubMed evidence, and IEDB IDs — e.g. P04637 (TP53) has 62 mapped epitopes. Use this over EBIProteins_get_antigen when the question is "has this region actually been shown to trigger an immune response" rather than "does this region look antigenic by sequence pattern" — relevant for vaccine/antibody design questions that land in this skill via a PTM-and-antigenicity angle.

Evidence Grading

Tier Criteria
T1 PTM at validated active/binding site with functional data
T2 PTM in structured domain with ProtVar annotation
T3 Correlation data only (mass spec detection)
T4 Predicted, no experimental validation

Tool Parameter Reference

Tool Key Params
iPTMnet_search operation="search", search_term, role
iPTMnet_get_ptm_sites operation="get_ptm_sites", uniprot_id
iPTMnet_get_proteoforms operation="get_proteoforms", uniprot_id
iPTMnet_get_ptm_ppi operation="get_ptm_ppi", uniprot_id
ELM_get_instances operation="get_instances", uniprot_id, motif_type
ELM_list_classes operation="list_classes"
MassIVE_search_datasets page_size, species
EBIProteins_get_mutagenesis / _get_proteomics_ptm / _get_antigen / _get_coordinates / _get_proteomics_peptides / _get_hpp_peptides / _get_domains_sites / _get_molecule_processing / _get_structural_features / _get_epitopes accession (UniProt)
EBIProteins_get_features accession, category (default DOMAINS_AND_SITES)
EBIProteins_get_coordinate_mapping accession (UniProt)
EBIProteins_get_proteins_by_genomic_loc taxonomy (default 9606), location ("chr:pos") or chromosome+position
EBIProteins_get_variation accession, optional source_type, disease_only
EBIProteins_get_variation_by_dbsnp dbsnp_id (rsID)
EBIProteins_get_variation_by_hgvs hgvs (genomic, NC_...:g....)

Critical: All iPTMnet and ELM tools require operation as first parameter (SOAP-style). EBI Proteins tools take a plain accession/dbsnp_id/hgvs string, no operation wrapper.

Fallbacks

Situation Fallback
Not in iPTMnet UniProt PTM/processing annotations
No PTM-PPI data STRING general PPI
No ProtVar data UniProt domain annotations
No ELM data Proceed with iPTMnet/UniProt only
EBIProteins_get_rna_editing 404s Verified broken as of this writing (EBI-side, not a request bug) — skip, do not retry-loop on it

Limitations

  • iPTMnet biased toward well-studied proteins
  • Proteoform data covers observed combinations only
  • PTM-PPI: only PTM-specific evidence; more PPIs exist in STRING
  • EBIProteins_get_proteomics_ptm/_get_proteomics_peptides/_get_hpp_peptides are MS detection evidence, not curated PTM biology — a hit means "seen by mass spec," not "known functional PTM"
  • EBIProteins_get_rna_editing is currently non-functional (HTTP 404 from EBI on both of its own documented example accessions, confirmed live) — do not rely on it until re-verified