Milli q h2o

LC-MS/MS analysis was carried out using a nanoAcquity HPLC (Waters, Milford, MA) and a LTQ-Orbitrap XL mass spectrometer (Thermo Scientific). For each analysis, 5 μL of tryptic digest was loaded onto a 180 μm x 20 mm trap column packed with 5 μm Symmetry C18 resin (Waters) using solvent A (0.1% formic acid in Milli-Q H₂O [Millipore, Billerica, MA]) followed by separation on a 75 μm x 250 mm analytical column packed with 1.7 μm BEH130 C18 resin (Waters). Peptides were eluted with either a 4 or 8 hour chromatographic gradient using solvent A and solvent B (0.1% formic acid in acetonitrile). For the 4 hour gradient, peptides were eluted using 5–28% B over 170 minutes, 28–50% B over 50 minutes, 50–80% B over 10 minutes, constant 80% B for 10 minutes, and 80–5% B over 5 minutes. For the 8 hour gradient, peptides were eluted using 5–28% B over 350 minutes, 28–50% B over 150 minutes, 50–80% B over 20 minutes, constant 80% B for 10 minutes, and 80–5% B over 5 minutes. A 30 minute blank gradient was run in between each sample injection to minimize carryover. Full scans were carried out from 400–2000 m/z with 60,000 resolution. MS2 data were acquired in data-dependent mode of the top six most intense ions with dynamic exclusion enabled for 60 s, monoisotopic precursor selection enabled, and single charged ions rejected.

All solutions were prepared using nanopure Milli-Q H₂O (Millipore). H₂O₂ and HOCl concentrations were quantified spectrophotometrically (H₂O₂, ε_{240 nm} 43.6 M⁻¹ cm^{−1 56 (link)}; HOCl (at pH 12), ε_{292 nm} 350 M⁻¹ cm^{−1 57 (link)}). pH control of reaction mixtures was achieved by using 0.1 M sodium phosphate buffer, pH 7.4. All chemicals were obtained from Sigma (Sigma-Aldrich) unless otherwise stated. Human polymorphonuclear leukocyte-derived MPO was purchased from Planta Natural Products (Vienna, Austria).

Caution! Curium is a radioactive element of high radiotoxicity requiring special precautions for handling radioactive materials, and all studies were conducted in a laboratory dedicated to actinide research.All chemicals were used as obtained. Stock solutions were prepared by weighing and dissolving appropriate amounts of EuCl₃·6H₂O (99.99%, Sigma-Aldrich, Taufkirchen, Germany), Na₂HNTA (≥99%, Sigma-Aldrich), H₄EDTA (≥99%, Roth, Karlsruhe, Germany), and H₄EGTA (≥99%, Roth) in NaCl (99.5%, Roth) containing Milli-Q H₂O (18.2 MΩ cm, Millipore, Merck, Darmstadt, Germany) and D₂O (99.98% D, Deutero, Kastellaun, Germany) aqueous solutions. pH was adjusted with HCl (1.0 M, 0.1 M, and 0.01 M) and NaOH (1.0 M, 0.1 M, and 0.01 M) in D₂O solutions, likewise, DCl and NaOD (both >99% D, Deutero), using a pH meter (inoLab pH 730, Xylem, Weilheim, Germany) equipped with a pH electrode (SCHOTT, BlueLine, SI Analytics, Mainz, Germany).
²⁴⁸Cm was obtained from the transplutonium element production facilities at Oak Ridge National Laboratory, Oak Ridge, TN, USA. Appropriate dilutions were made from a 295 µM Cm(ClO₄)₃ stock solution.

Sample preparation was performed in the clean room facilities of the MAGIC Laboratories, Imperial College London. The mineral acids used in this study, 15.4 mol L⁻¹ HNO₃, 5.8 mol L⁻¹ HCl and 28 mol L⁻¹ HF, were prepared by sub-boiling distillation in Teflon or quartz glass stills from AnalaR grade stock acids. All acid dilutions used Milli-Q H₂O from a Millipore purification system. Samples were stored in acid-cleaned Savillex Teflon vials.

The starting materials, 1,3-dicyanobenzene
(1,3-DCB), zinc chloride, and iron(II) acetate (all purchased from
Aldrich), were mixed in a glovebox in 1:1 DCB/ZnCl₂ and
0.16 Fe(OCH₃)₂/DCB molar ratios. The mixture
was ground, placed into a Pyrex vial, and sealed under vacuum. The
vial was heated from room temperature (r.t.) to 400 °C at 3 °C/min,
kept at 400 °C for 46 h, and cooled to r.t. A black monolith
(poly-1,3-DCB) was obtained and ball-milled in a planetary ball mill
for 60 min. The recovered solid was thermally treated under a temperature
program consisting of a heating ramp from r.t. to 900 °C at 20
°C/min, dwelling at 900 °C for 30 min under NH₃/N₂ flows of 28.1 and 24.2 mL/min, respectively, and cooling
to r.t. under a N₂ atmosphere. The catalyst obtained is
referred to as 1HT-1,3DCB. To remove the unstable Fe phases, we subjected
1HT-1,3DCB to acid leaching in 0.5 M H₂SO₄ at
60 °C for 4 h and washed it with Millipore Milli-Q H₂O until the pH of the water obtained was ca. 6. Finally, the material
was thermally treated following the thermal treatment protocol previously
defined. The catalyst obtained is labeled 2HT-1,3DCB.

The prostatic tissue (10–15 mg) was added to 500 μL of milliQ H₂O (Millipore, Billerica, MA, USA) and homogenized at 20,000 rpm by an ULTRATURRAX T25 (IKA, Staufen, Germany). After the addition of 50 μL of α-tocopherol acetate (2 mg/dL in methanol), the mixture was vortexed and subsequently added with 1 mL hexane and extensively vortexed. After centrifugation (4000 rpm, 10 min), 800 μL of the organic phase was transferred in sterile tubes and dried by vacuum centrifugation. The pellet was vortexed, suspended in 100 μL of methanol and injected into the chromatographic column. The HPLC separation was performed according to the protocol described above.