Use of mass spectrometry to probe the nucleophilicity of cysteinyl residues of prolyl hydroxylase domain 2.
Prolyl hydroxylase domain 2 (PHD2) plays an important role in hypoxic sensing in humans. Here we report studies on the reactivity of cysteinyl residues of the catalytic domain of PHD2 using an approach in which nondenaturing electrospray ionization-mass spectrometry (ESI-MS) analyses were combined with the use of a thiol library and residue substitution. Among the seven cysteinyl residues of the PHD2 catalytic domain, Cys201 was found to be predominantly modified by thiols or N-ethylmaleimide. Selective modification of Cys201 was further demonstrated with methanethiosulfonate, a spin-labeled probe. The modified PHD2 will be useful in electron paramagnetic resonance studies on PHD2. The results demonstrate the use of a combined library/residue substitution/ESI-MS approach for analyzing residue reactivity.
Mecinović
Jasmin
J
Chemistry Research Laboratory and Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK.
Chowdhury
Rasheduzzaman
R
Flashman
Emily
E
Schofield
Christopher J
CJ
D000477:Alkylating Agents; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry*; D004926:Escherichia coli; D005033:Ethylmaleimide; D015870:Gene Expression; D006801:Humans; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D008698:Mesylates; D011393:Procollagen-Proline Dioxygenase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013113:Spin Labels; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents
D000477:Alkylating Agents; D008698:Mesylates; D011994:Recombinant Proteins; D013113:Spin Labels; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C074502:methanethiosulfonate; C490190:EGLN1 protein, human; D011393:Procollagen-Proline Dioxygenase; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D003545:Cysteine; D005033:Ethylmaleimide