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PXD013112-1

PXD013112 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleLack of activity of recombinant HIF prolyl hydroxylases (PHDs) on reported non-HIF substrates
DescriptionHypoxia 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.
HostingRepositoryPRIDE
AnnounceDate2019-08-30
AnnouncementXMLSubmission_2019-08-30_07:54:05.xml
DigitalObjectIdentifierhttps://dx.doi.org/10.6019/PXD013112
ReviewLevelPeer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportSupported dataset by repository
PrimarySubmitterMatthew Cockman
SpeciesList scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationListGluGlu; 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
InstrumentQ Exactive
Dataset History
RevisionDatetimeStatusChangeLog Entry
02019-03-18 02:15:18ID requested
12019-08-30 07:54:06announced
Publication List
Dataset with its publication pending
Keyword List
curator keyword: Biological
submitter keyword: Human, prolyl hydroxylation, PHD enzyme, in vitro, LC-MSMS
Contact List
Peter John Ratcliffe
contact affiliationFrancis Crick Institute London. NW1 1AT. United Kingdom
contact emailpeter.ratcliffe@crick.ac.uk
lab head
Matthew Cockman
contact affiliationFrancis Crick Institute
contact emailmatthew.cockman@crick.ac.uk
dataset submitter
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