Rapigest sf solution

The suspensions containing the total protein samples from bacterial lysate of E. coli (ATCC 8739), and magainin I-susceptible and -resistant E. coli strains were digested with trypsin enzyme (Promega) for acquisition in a nanoUPLC-MS^E. Briefly, 100 μg of each protein sample was mixed with 50 mM ammonium bicarbonate and 0.2% RapiGest SF solution (Waters). The mixture was homogenized and incubated at 80 °C, for 15 min. Then, 100 mM dithiothreitol was added and the samples were incubated at 60 °C, for 30 min. After that, the samples were briefly centrifuged and 300 mM iodoacetamide was added, remaining incubated at room temperature, for 30 min and protected from light. After time incubation, trypsin enzyme (Promega) was added in a 1:100 ratio (v/v) and the samples were incubated at 37 °C, for 16 h for time digestion. For surfactant hydrolysis and precipitation, 5% TFA was added at 37 °C, for 90 min. The samples were centrifuged at 14,000 g, 6 °C, for 30 min and the supernatants were recovered and lyophilized. After that, the samples were re-suspended in 190 µL of 20 mM of ammonium formate (Sigma-Aldrich). Also, 10 µL of MassPREP^TM digestion standard Phosphorylase b (Waters) was added (stock 1pmol.μL⁻¹) as a standard of protein digestion (final concentration of 50 fmol.μL⁻¹). The sample preparation was performed in triplicate.

Each fat depot specimen from each animal was prepared and analyzed independently (i.e., not pooled). Retroperitoneal fat pads were removed and homogenized in 1 ml of lysis buffer, containing: 8 M urea, 75 mM NaCl, 1 M Tris, and complete Mini Protease Inhibitor Cocktail Tablets (Roche Diagnostics, USA). Samples were then centrifuged at 19,000 × g for 30 min at 4°C. Total protein concentration was determined using the Lowry method (Lowry et al., 1951 (link)). As previously described (Pedroso et al., 2017 (link)), aliquots corresponding to 2,000 µg of total protein were transferred to Amicon Ultra-4 Centrifugal 3,000 NMWL filter devices (Merck Millipore), for buffer exchange into 50 mM NH₄HCO₃. Next, the protein concentration was measured again, and 200 µg of total protein was added to 25 µl of 0.2% RapiGest SF solution (Waters, USA) and incubated for 15 min at 80°C. Samples were then reduced with 5 mM DTT for 30 min at 60°C, and alkylated with 10 mM iodoacetamide, in the dark for 30 min at room temperature. Proteins were digested with trypsin Gold (Promega, USA) at a protease/protein ratio of 1:100 (w/w), overnight at 37°C. Digestions were terminated by adding 10 µl of 5% trifluoroacetic acid (TFA) for 90 min at 37°C. Samples were centrifuged at 19,000 × g for 10 min at 4°C, and the supernatants were collected. The final protein concentrations were typically around 2 µg/µl.