Tributyl phosphate

p-Toluenesulfonyl
chloride (97%), p-toluenesulfonyl amide (97%), lithium
hydroxide monohydrate, potassium permanganate, calcium chloride, N-methyl-2-pyrrolidone (NMP, anhydrous, 99.8%), pyridine
(anhydrous, 99.8%), lithium aluminum hydride (95%), tetrahydrofuran
(THF, anhydrous, 99.8%), lithium bis(trimethylsilyl) amide solution
(1 M in THF), tributyl phosphate (≥99%), and titanium(IV) isopropoxide
(97%) were purchased from Sigma-Aldrich. Aluminum nitrate (Al(NO₃)₃ × 9H₂O), 3-(aminopropyl)triethoxysilane,
1-propanol, and propionic acid were acquired from Merck. 4,4-(Hexa-fluoroisopropylidene)dianiline
(98%) was obtained from TCI Europe, poly(vinylidene difluoride-co-hexafluoropropylene)
(PVdF-HFP, Kynar FLEX LBG) from Arkema, and lithium
nitrate (LiNO₃ × xH₂O)
from Alfa Aesar. Concentrated hydrochloric acid, methanol, dimethyl
sulfoxide (DMSO), and triphenyl phosphite were acquired from VWR.
Ethylene carbonate, propylene carbonate, and NMC622 were purchased
from BASF. Carbon black (Super C65) was obtained from Imerys Graphite
& Carbon and polyvinylidene difluoride (PVdF, Solef 5130) from
Solvay. Prior to use, calcium chloride was dried at 180 °C under
reduced pressure (10^–3 mbar) for 48 h.

Amorphous tricalcium phosphate NPs with 2 % of copper (CuTCP) or without copper (aCaP with Ca:P = 1.5 [25 ]) were prepared by flame spray synthesis [26 ] and a size of ∼22 nm. For aCaP NPs, calcium-2-ethyl-hexanoic salt (synthesized by calcium hydroxide from Riedel de Haen, Ph. Eur. and ethylhexanoic acid from Sigma-Aldrich, St. Louis, USA) and tributyl phosphate (98% Sigma-Aldrich) were used. The CuO NPs were received as Copper nanopowder from Sigma Aldrich (40–60 nm (SAXS)). NPs were characterized with X-ray diffraction (XRD).

A lysis buffer composed of 7 M urea, 2 M thiourea, 4% w/v 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (Sigma, St. Louis, MO, USA), 20 mM tributyl phosphate (Sigma, St. Louis, MO, USA), and 0.2% Bio-lyte (pH 3–10) plus a protease inhibitor cocktail (Roche Diagnostics Ltd, Mannheim, Germany) were used to re-suspend and disrupt the isolated cells. The lysate was then treated with DNAse I (Qiagen, Hilden, Germany) and RNAse A (Thermo Fisher Scientific, Waltham, MA, USA) at final concentrations of 1 mg/mL and 0.25 mg/mL, respectively. The protein solution was separated from the cell debris by centrifugation (12,000 × g, 4 °C, 5 min). A Ready Prep 2-D Cleanup Kit (Bio-Rad Laboratories, Hercules, CA, USA) was used for further purification of the crude protein, which was then subjected to a reductive alkylation reaction. The protein concentration was measured with a 2-D Quant Kit (GE Healthcare, San Diego, CA, USA). Afterward, trypsin digestion and iTRAQ labeling were performed according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA, USA). Briefly, 100 μg of total protein per sample was reduced and alkylated, then digested overnight with trypsin (Promega, Madison, WI, USA) at 37 °C. It was labeled with iTRAQ-reagents (Applied Biosystems) as follows: 7 days, iTRAQ reagent 115; 14 days, iTRAQ reagent 116; and 21 days, iTRAQ reagent 117.