Total serum and liver BAs were extracted and analyzed by Thermo Finnigan Ultra Performance Liquid Chromatography (UPLC) system coupled with a Thermo Finnigan LTQ XL Ion Trap Mass Spectrometer (Thermo Fisher Scientific). Detailed protocols were described in our previous publications [25 (link), 32 (link)]. The percent composition of specific BAs in the serum or liver was calculated. In total, 21 BA species were evaluated, including: cholic acid (CA), taurocholic acid (TCA), glycocholic acid (GCA), chenodeoxycholic acid (CDCA), taurochenodeoxycholic acid (TCDCA), glycochenodeoxycholic acid (GCDCA), α-muricholic acid (α-MCA), β-muricholic acid (β-MCA), ω-muricholic acid (ω-MCA), tauromuricholic acid (TMCA), deoxycholic acid (DCA), taurodeoxycholic acid (TDCA), glycodeoxycholic acid (GDCA), ursodeoxycholic acid (UDCA), tauroursodeoxycholic acid (TUDCA), glycoursodeoxycholic acid (GUDCA), hyodeoxycholic acid (HDCA), taurohyodeoxycholic acid (THDCA), lithocholic acid (LCA), taurolithocholic acid (TLCA), and glycolithocholic acid (GLCA).
Thermo finnigan ltq xl ion trap mass spectrometer
Manufactured by Thermo Fisher Scientific
The Thermo Finnigan LTQ XL Ion Trap Mass Spectrometer is a high-performance analytical instrument designed for the detection and identification of chemical compounds. It utilizes ion trap technology to capture, isolate, and analyze ions based on their mass-to-charge ratio. The system provides accurate mass measurements and tandem mass spectrometry capabilities for the structural elucidation of molecules.
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2 protocols using thermo finnigan ltq xl ion trap mass spectrometer
1
Comprehensive BA Profiling by UPLC-MS
2
Antibody Deglycosylation and Proteolytic Digest
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Antibodies were reduced with 10 mM dithiothreitol, 4 M guanidine HCl, 57 mM Tris-HCl pH 8.3 at 55 C for 30 min. After alkylating in 22 mM iodoacetic acid for 15 min at room temperature, the protein was desalted using a Biospin-6 gel filtration cartridge (BioRad) and then deglycosylated for 2 h at 37 C using PNGaseF (New England BioLabs). Endoproteinase digestion was carried out using 10% trypsin (Promega) in 50 mM Tris-HCl pH 7.5, 4 M urea at 37 C for 4 h. Peptides were separated by RP-HPLC on 2 Waters BEH300 columns in series (1.7 mM particle, 2 Â 100 mm) at a flow rate of 0.2 mL/min and an acetonitrile gradient of 0.3%/min. Peptides were detected by UV (215 nm) and by MS and MS/MS on a Thermo Finnigan LTQ XL ion trap mass spectrometer (Thermo Scientific, Waltham, MA) set to carry out 1 full survey scan followed by 3 CID-MS 2 scans.
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