2 2 2 trifluoroethanol

Following injection into the trapping column (µPAC trapping column, Pharma Fluidics, Gent, Belgium), the peptides were separated by nano-reverse-phase (µPAC, 200 cm separation column, Pharma Fluidics, Gent, Belgium) using an UltiMate3000 nano rapid separation liquid chromatography HPLC (Thermo Fisher, Germering, Germany) separation system. Both, the trap- and separation columns were operated at 50 °C, and the UV peptide detection at 214 nm served as quality control for the HPLC separation. The samples were loaded onto the trap column using a loading solvent of 2% acetonitrile (ACN, VWR, Vienna, Austria) in an aqueous mix of 0.1% trifluoroacetic acid (TFA)/0.01% heptafluorobutyric acid (HFBA), both purchased from Sigma-Aldrich, Vienna, Austria, at 30 μL/min and precooled to 3 °C [36 (link)]. Nano separation was performed in gradient mode at 600 nL/min. A user defined injection program was used for sample injection and additional injector and trap column wash. Every sample injection was followed by two blank runs with injections of 2,2,2-trifluoroethanol (Alfa-Aeser, Vienna, Austria) for the removal of possible sample remains in the injector or on the trap column and for the prevention of carryover in the separation system. In order to perform the label-free quantitation (LFQ), equimolar amounts of peptides were injected.

Bovin serum albumin, catalase from bovine liver lyophilised powder (2000–5000 units/mg protein), ethanol, hydrogen peroxide solution (30% w/w in H₂O), methanol, polyvinylpyrrolidone (55 kDa), potassium hydroxide, Purpald^®, sodium chloride, sodium citrate tribasic dihydrate, and sodium phosphate monobasic monohydrate were supplied by Sigma-Aldrich (Steinheim, Germany). Citric acid, L-arginine monohydrochloride, dextran (~70 kDa), and 2,2,2-trifluoroethanol (>99%) were provided by Alfa Aesar (Heysham, UK). Ethylene diaminetetraacetic acid disodium salt dihydrate (EDTA), sodium phosphate dibasic dihydrate, aqueous formaldehyde solution (35 wt%) and hydrochloric acid (36.5–38% in water) were obtained from Honeywell (Seelze, Germany). Potassium periodate was purchased from Scientific Laboratory Suppliers Ltd. (Nottingham, UK). Ficoll PM70 (polysucrose 70) was obtained from Cytiva (Uppsala, Sweden). The Bradford 5× reagent was obtained from Serva (Heidelberg, Germany) and a microBCA protein assay kit was sourced from ThermoFisher (Rockford, IL, USA). Super refined Polysorbate 20^® and super refined Polysorbate 80^® were obtained from Croda (Goole, UK). α,α-Trehalose dihydrate was purchased from Pfanstiehl (Waukegan, IL, USA).

Gas chromatography was carried out in a Hewlett Packard 5890 and 7890 series II Gas Chromatograph using a column Zebron ZB-Wax 30 m × 0.25 mm × 0.25 μm for the determination of the results of the reaction of glycidol with methanol, and for the rest of the alcohols, a column of phenyl silicone 5.5% (Zebron ZB-5HT Inferno 30 m × 0.25 mm × 0.25 μm) was used. Helium was used as a carrier gas, and the detection was done with a flame ionization detector (FID).
Glycidol, diglyme, Dowex 50WX2, K10 Montmorillonite, Aquivion PW79S and Amberlyst 15 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol and isopropanol, were purchased from Scharlab (Barcelona, Spain). Butanol, 2,2,2-trifluoroethanol and Nafion NR50 were purchased from Alfa Aesar (Haverhill, MA, USA).