Glycochenodeoxycholic acid gcdca

The hSOD1 aggregate preparation was vortexed for 2 min and then 10 µl aliquots were incubated with or without different concentrations of trypsin (10, 100 and 1000 µg/ml; Sigma-Aldrich); proteinase K (10, 50 and 250 µg/ml; Thermo Fisher Scientific); sodium dodecyl sulfate (SDS; 0.3, 1, 3 and 10 g/L; Sigma-Aldrich); or glycochenodeoxycholic acid (GCDCA; 8 mM; Sigma-Aldrich) in a total volume of 50 µl of PBS for different time intervals (0, 0.5, 1, 2, 4 and 6 h) at 37 °C using a shaker (IKA). GCDCA incubation was performed in the presence of the Complete EDTA-free protease inhibitor cocktail (Roche Diagnostics). Proteolysis with proteinase K was terminated by the addition of phenylmethylsulphonylflouride (PMSF; 20 and 100 mM; Sigma-Aldrich). After completed incubation, each sample reaction was immediately attenuated by the addition of 500 µl of water as a diluent, snap-frozen in liquid nitrogen and stored at − 80 °C. Frozen samples were thawed in a water bath at 25 °C for 2 min and then centrifuged at 25,000 g for 30 min at 4 °C and the supernatants were transferred to new tubes. The pellets were washed by suspension in 1 ml PBS and centrifuged again at 25,000 g for 30 min at 4 °C. The amount of hSOD1 in both supernatant and pellet was determined by western blotting.

CA was purchased from Alfa Aesar (Ward Hill, MA), taurocholic acid (TCA) from Acros Organics (Pittsburgh, PA) and glycocholic acid (GCA) from Pfaltz & Bauer (West Chester, PA). CDCA, glycochenodeoxycholic acid (GCDCA), taurochenoxycholic acid (TCDCA), deoxycholic acid (DCA), glycodeoxycholic acid (GDCA), taurodeoxycholic acid (TDCA) and deoxy-2,2,4,4-d4 acid (DCA-d4) were purchased from Sigma-Aldrich (St. Louis, MO). Cholic-2,2,4,4-d4 acid (CA-d₄), glycocholic-2,2,4,5-d4 acid (GCA-d₄), chenodeoxycholic-2,2,4,4-d4 acid (CDCA-d4) and glycochenodeoxycholic-2,2,4,4-d4 acid (-GCDCA-d4) were purchased from C/D/N ISOTOPES Inc. (Quebec, Canada). Taurocholic 2,2,3,4,4-d₅ acid (TCA- d₅) and taurochenodeoxycholic-2,2,3,4,4-d₅ acid (TCDCA-d₅) were purchased from Toronto Research Chemicals Inc. (Ontario, Canada). HPLC grade methanol, acetonitrile, water and formic acid were purchased from Honeywell Burdick & Jackson (Muskegon, MI). Charcoal stripped serum was purchased from Bioreclamation (Westbury, NY).