Trifluoracetic acid

Polydimethylsiloxane, hydroxy-terminated (PM 550, PDMS-OH), cyanopropyltriethoxysilane (CPTEOS), and dichloromethane (DCM) were all obtained from Sigma-Aldrich Co. Trifluoracetic acid (TFA), HPLC grade methanol, HPLC grade acetonitrile (ACN), and HPLC grade water, were provided by J.T. Baker. Sodium hydroxide (NaOH) from Fluka Analytical and hydrochloric acid (HCl) from Meyer were also used. All reagents were at least 98% pure.

Cultures of B. pertussis were pelleted by centrifugation (10,000× g, 4 °C, 10 min) to separate cell pellets and culture supernatants. Cells were resuspended in TEAB digestion buffer (100 mM Triethylammonium bicarbonate, pH 8.5, 2% sodium deoxycholate) and lysed by sonication. For analysis of supernatant fractions, supernatants were filtered through 0.22-μm filters and precipitated with 10% (w/v) trichloracetic acid (Sigma) overnight at 4 °C. Precipitated proteins were collected by centrifugation (14,000× g, 4 °C, 20 min), washed with 80% acetone (w/v) and finally dissolved in TEAB digestion buffer. Protein concentrations were determined using BCA protein assay kit (Thermo Fischer Scientific) and 20 µg of protein per sample were used for protein analysis. Cysteines were reduced with M Tris(2-carboxyethyl)phosphine (60 °C for 60 min) and blocked with 1M methyl methanethiosulfonate (10 min, room temperature). Samples were digested with trypsin (trypsin to protein ratio 1:20) at 37 °C overnight. Digestion of samples was stopped by addition of trifluoracetic acid (Sigma) to a final concentration of 1% (v/v). SDC was removed by extraction with ethylacetate [68 (link)] and peptides were desalted on C18 column (Michrom Bio, Auburn, CA, USA).

Naftifine hydrochloride was purchased from ChemicalPoint (Deisenhofer, Germany). Ethanol 96% (Vilniaus degtinė, Vilnius, Lithuania), butyl acetate and ethyl acetate were obtained from Sigma-Aldrich Chemie GmbH (Steinheim, Germany) and used as a solvent system. Triacetin which was used as plasticizer was kindly supplied by Lanxess (Leverkusen, Germany). Film-forming polymer Eudragit RL100 was kindly gifted by Evonik Industries AG (Essen, Germany). Salicylic acid (Alfa Aesar, Karlsruhe, Germany), glycerol (Applichem, Darmstad, Germany); 1.2-propandiol, polyethylenglycol 400, polyethylenglycol 1500, urea was purchased from Roth (Karlsruhe, Germany) and used as chemical enhancers. Tween 60, Tween 40, citric acid monohydrate, sodium carbonate, acetone, benzoic acid, and methanol were used as enhancers and obtained from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Thioglycolic acid was kindly gifted by Merck Group (Darmstadt, Germany). Acetonitrile and trifluoracetic acid for chromatography analysis were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany).