Q exactivetm

The methods of protein extraction and trypsin digestion were performed according to the protocol reported in Sun et al. (2017) (link) with slight modifications. The gradient was comprised of an increase from 6 to 22% of solvent B (0.1% FA in 98% ACN) for 40 min, from 22 to 35% over 12 min, climbing to 80% over 4 min, and then holding at 80% for the last 4 min. A constant flow rate of 300 nL/min was maintained on an EASY-nLC 1000 UPLC system, and the resulting peptides were analyzed by a Q Exactive^TM plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific).
The peptides were subjected to Nanospray ionization (NSI) source followed by tandem mass spectrometry (MS/MS) in Q Exactive^TM plus (Thermo) coupled with an online Ultra Performance Liquid Chromatography (UPLC). For MS scans, the m/z scan range was 350 to 1800. Fixed first mass was set as 100 m/z.

The tryptic peptides were fractionated through high pH reverse-phase HPLC using Thermo Betasil C18 column (5 μm particles, 10 mm ID, 250 mm length). Then the peptides were separated into 60 fractions by a gradient of acetonitrile (8–32%, pH 9.0). Finally, the peptides were combined into 6 fractions and dried by a vacuum-type centrifuge. LC-MS/MS Analysis and Database Search were conducted according to the previously published method (Huang et al., 2020 (link)). Briefly, we subjected the peptides to an NSI source, followed by MS/MS in Q ExactiveTM (Thermo Fisher Scientific, Waltham, MA, United States) coupled with UPLC online. Maxquant search engine (v.1.5.2.8) was used to process the resulting MS/MS data. Tandem mass spectra was searched with the uniprot database. Trypsin/P was specified as cleavage enzyme, and we allowed up to four missing cleavages. About 20 ppm was set for the mass tolerance for precursor ions in First search, and 5 ppm was used in Main search. About 0.02 Da is set for the mass tolerance for fragment ions. FDR was adjusted to <1%.

Feces, collected on day 9 of the intervention, were stored at −70°C, and the bile acids were detected by the Beijing Bio-Tech Pack Technology Company Ltd. (Beijing, China). Briefly, feces (100–200 mg) were suspended in 1 mL methanol and extracted by vortexing for 60 min, following centrifugation at 13,200 r/min for 10 min. A total of 500 μL of supernatant was centrifuged and volatilized by freeze-drying under vacuum, followed by the addition of 150 μL methanol to the Eppendorf tube to dissolve the sediment. The supernatant was detected on a liquid chromatograph (LC, UltiMate 3000 UHPLC, Thermo Scientific^TM, United States) and mass spectrometer (MS, Q Exactive^TM, Thermo Scientific^TM, United States). The concentration of the bile acids was calculated according to the peak areas of the bile acids and external standards.

The samples were fractionated by high pH reverse-phase High Performance Liquid Chromatography (HPLC). Briefly, peptides were dried by vacuum centrifugation. Then the Peptides were dissolved in 0.1% formic acid and directly loaded onto a reversed-phase precolumn (Acclaim PepMap 100, Thermo Scientific). Peptides were separated using a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific) and analyzed by Q Exactive^TM hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific).
The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q Exactive^TM (Thermo Fisher Scientific) coupled online to the HPLC. Intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using NCE setting as 32. Ion fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 2E4 in the MS survey scan with 30.0 s dynamic exclusion. The electrospray voltage was 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the ion trap. 5E4 ions were accumulated for generation of MS/MS spectra. For MS scans, the m/z scan range was 350 to 1800. Fixed first mass was set as 100 m/z.