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PXD013112 is an original dataset announced via ProteomeXchange.

Dataset Summary

  • Title: Lack of activity of recombinant HIF prolyl hydroxylases (PHDs) on reported non-HIF substrates
  • Description: Hypoxia Inducible Factor (HIF) prolyl hydroxylase domain (PHD) enzymes catalyse the posttranslational hydroxylation of conserved prolyl residues in the alpha-subunit of the HIF transcription factor. These modifications, which promote the degradation of HIF-alpha subunits by the pVHL E3 ligase complex and impart oxygen-dependent regulation of the HIF transcriptional response, have been extensively characterised at the molecular, cellular and organismal level. Since the discovery of the PHDs, a range of less well-characterised non-HIF substrates have been reported with the potential to confer oxygen-sensitivity to a diverse range of cellular processes. We sought to systematically compare all of the reported non-HIF substrates for their ability to support PHD-catalysed hydroxylation. We performed a comprehensive series of in vitro hydroxylation assays reacting synthetic peptides and full-length protein substrates generated by in vitro transcription and translation with purified recombinant enzyme preparations. Prolyl hydroxylation was assayed directly by mass spectrometry and radiochemical assay for hydroxyproline. Both methods enabled quantitative appraisal of enzyme-catalysed hydroxylation, with liquid chromatography mass spectrometry methods employing NMR-quantified peptide standards to calibrate retention time signatures and determine ionisation efficiencies for each target peptide. Using these approaches we assayed hydroxylation on 23 different proteins. Surprisingly, we did not detect measurable hydroxylation on any of the reported non-HIF substrates using either method. In contrast, control assays with HIF1-alpha substrate supported high stoichiometry (typically >90%) hydroxylation. Our findings suggest that PHD substrates are more restricted than has been reported.
  • HostingRepository: PRIDE
  • AnnounceDate: 2019-08-30
  • AnnouncementXML: Submission_2019-08-30_07:54:05.xml
  • DigitalObjectIdentifier: http://dx.doi.org/10.6019/PXD013112
  • ReviewLevel: Peer-reviewed dataset
  • DatasetOrigin: Original dataset
  • RepositorySupport: Supported dataset by repository
  • PrimarySubmitter: Matthew Cockman
  • SpeciesList: scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
  • ModificationList: GluGlu; Crotonyl; CAMthiopropanoyl; Decanoyl; GlycerylPE; LeuArgGlyGly; Methylpyrroline; Delta:H(2)C(5); Delta:S(-1)Se(1); Met->Hsl; Diethylphosphate; Guanidinyl; DAET; BHTOH; LRGG+dimethyl; Nethylmaleimide+water; LRGG+methyl; Ethylphosphate; ISD_z+2_ion; Oxidation; Piperidine; Met->Hpg; Delta:H(4)C(2); O-pinacolylmethylphosphonate; BITC; Didehydroretinylidene; 4-ONE; Quinone; Cation:Na; maleimide3; Sulfo; Deoxy; Malonyl; Methyl+Deamidated; HPG; GluGluGlu; Formyl; Carbamyl; Arg->Orn; Delta:H(2)C(3)O(1); Phosphoguanosine; Cation:Mg[II]; PhosphoUridine; Met->Aha; Tyr->Dha; NDA; Amino; Dimethyl; Cation:Cu[I]; Hypusine; Pro->Pyrrolidone; Crotonaldehyde; NeuAc; Dethiomethyl; Thioacyl; Lys->AminoadipicAcid; Pro->pyro-Glu; Phospho; Acetyl; Palmitoyl; Cation:Ni[II]; Nitro; Methylthio; dichlorination; Carboxymethyl; 4-ONE+Delta:H(-2)O(-1); HexNAc; Carbamidomethyl; Cation:Ca[II]; 3sulfo; HNE+Delta:H(2); trifluoro; Puromycin; Glu->pyro-Glu; Arg->Npo; GluGluGluGlu; Carboxyethyl; Hydroxymethyl; CAF; EDT-maleimide-PEO-biotin; Delta:H(2)C(2); BHT; phosphoRibosyl; Isopropylphospho; LG-pyrrole; LG-Hlactam-R; Phosphopropargyl; Delta:H(4)C(3); Didehydro; Cytopiloyne+water; SMA; Arg2PG; Diisopropylphosphate; Lys->Allysine; Heme; LG-anhyropyrrole; Amidine; ethylamino; Hydroxyheme; Ethyl+Deamidated; Benzoyl; glycidamide; HNE; Bacillosamine; Hex; Phosphopantetheine; SPITC; Bromo; Methylphosphonate; Delta:H(4)C(2)O(-1)S(1); DFDNB; Propionamide; Deamidated; sulfo+amino; Trp->Oxolactone; Methylamine; azole; DTT_ST; CHDH; Myristoyl; Cation:K; PyridoxalPhosphate; Argbiotinhydrazide; Octanoyl; Gln->pyro-Glu; Menadione-HQ; NA-LNO2; Chlorination; Trimethyl; Fluoro; Ammonia-loss; HydroxymethylOP; Diphthamide; AEC-MAEC; Ethanedithiol; Carboxy; PET; FMN; Ethanolamine; LG-lactam-R; Palmitoleyl; Hydroxytrimethyl; PyMIC; Trp->Hydroxykynurenin; MG-H1; GlyGly; 3-phosphoglyceryl; glucosone; LG-anhydrolactam; Thiophospho; 3-deoxyglucosone; Sulfide; FMNH; Ammonium; Menadione; NA-OA-NO2; Delta:H(5)C(2); Lipoyl; HCysThiolactone; Met->Hse; Cytopiloyne; Amidated; Diethyl; Ethanolyl; Cation:Zn[II]; Propargylamine; PEITC; cGMP; BisANS; ADP-Ribosyl; LG-Hlactam-K; 2HPG; Cation:Ag; Phosphoadenosine; Biotin-PEO-Amine; cGMP+RMP-loss; Glycerophospho; MercaptoEthanol; Cation:Li; Ethyl; GPIanchor; O-Isopropylmethylphosphonate; Cation:Fe[II]; FTC; Methyl; AEBS; Pyridylacetyl; Dihydroxyimidazolidine; Methylmalonylation; HNE-Delta:H(2)O; Ethoxyformyl; TNBS; DEDGFLYMVYASQETFG; MolybdopterinGD; Trp->Kynurenin; Glu; Pro->Pyrrolidinone; Arg->GluSA; Dansyl; Iminobiotin; Iodo; thioacylPA; Nmethylmaleimide; DimethylpyrroleAdduct; G-H1; maleimide; O-Methylphosphate; Dehydrated; Biotin; Dioxidation; Carbofuran
  • Instrument: Q Exactive

Dataset History

RevisionDatetimeStatusChangeLog Entry
02019-03-18 02:15:18ID requested
12019-08-30 07:54:06announced

Publication List

  1. Dataset with its publication pending

Keyword List

  1. curator keyword: Biological
  2. submitter keyword: Human, prolyl hydroxylation, PHD enzyme, in vitro, LC-MSMS

Contact List

    Peter John Ratcliffe
    • contact affiliation: Francis Crick Institute London. NW1 1AT. United Kingdom
    • contact email: peter.ratcliffe@crick.ac.uk
    • lab head:
    Matthew Cockman
    • contact affiliation: Francis Crick Institute
    • contact email: matthew.cockman@crick.ac.uk
    • dataset submitter:

Full Dataset Link List

  1. Dataset FTP location
  2. PRIDE project URI
Repository Record List

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