Nh4 2fe 2 so4 2

Hydroxylation assays were performed in triplicate under normoxic conditions using liquid chromatography electrospray MS (LC-ESI-MS) (Figure 3, Figure 4). Conditions: 1 μm TgPhyA or DdPhyA, 100 μm full-length DdSkp1 or full-length TgSkp1 substrate, 50 μm (NH₄)₂Fe(II)(SO₄)₂ (Sigma-Aldrich), 1 mm sodium l-ascorbate (Sigma-Aldrich), and 500 μm 2-oxoglutarate disodium salt (Sigma-Aldrich) in HEPES (100 mm), pH 7.6. Mixtures were incubated at 37 °C for 1 h, or at specific time points for time course assays, and quenched using an equal volume of 1% (v/v) aqueous formic acid (Sigma-Aldrich). Reaction mixtures were analyzed using a Xevo G2-S Q-TOF mass spectrometer equipped with an electrospray source (Waters®) coupled to a Waters® ACQUITY UPLC System, unless otherwise specified. Instrument control and data processing were performed using MassLynx V4.1 software. An Aeris^TM 3.6-μm WIDEPORE C4 200 Å (Phenomenex) 4.6 × 50 mm column was used for separation.

Chemicals were from commercial sources
unless stated otherwise. 2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethanesulfonic
acid (HEPES, Apollo Scientific Limited, Code: BI8181), sodium chloride
(Fisher Bioreagents, Cat#: BP358-10), bovine serum albumin (BSA, PerkinElmer,
Stabilizer, 7.5% (DTPA-purified BSA), Cat: CR84-100), Tween 20 (Tween
20, Promega, ref#: H5151), disodium 2-oxoglutarate (Sigma, Cat#: K3752–100G),
(+)-sodium L-ascorbate (Sigma, Cat#: 11140-50G), (NH₄)₂Fe(II)(SO₄)₂ (Sigma-Aldrich, cat#: 215406-100G),
ethanediaminetetraacetate disodium salt dihydrate (EDTA, Sigma, cat#:
E5134-500G), ^5hmC-specific antibody (Active Motif Cat#:
39769), AlphaScreen beads (PerkinElmer, IgG Detection Kit (Protein
A), Cat#: 6760617M, lot: 1571768), Complete EDTA-free Protease Inhibitor
Cocktail (Roche Diagnostics Ltd.), ProxiPlate -384 (White, Shallow
384 well, Pinch bar design, part#: 6008280), and DMSO (Fisher Chemical,
code: D/4120/PB08). Water, LiChroSolv for LC-MS (VWR cat#: (1.15333.2500)),
acetonitrile (LiChroSolv for LC, VWR, cat#: 1.00030.2500). N-oxalylglycine disodium was prepared according to the reported
procedure.^{83 (link),84 (link)}