Ltq velos

The immunoprecipitated proteins were separated by electrophoresis on 5% and 12% SDS-PAGE gels and the separation gel was stained using Coomassie brilliant blue for Mass Spectrometry. All distinct bands in the lane of HuN4-F112 infection group and the gel at parallel areas in the lane of the control group were excised and subjected to LC-MS/MS. Briefly, gel pieces were distained with 30% acetonitrile/100 mM NH₄HCO₃ and freeze-dried. The gel pieces were reduced with 100 mM of DTT (56 °C, 30 min), followed by alkylation with 200 mM iodoacetamide (in the dark, 25 °C, 20 min). The gels were incubated with 100 mM NH₄HCO₃ and shrunk with acetonitrile again, and incubated with trypsin (2.5-10 ng/μL) for 20 h at 37 °C. Peptides were extracted with 60% acetonitrile/0.1% TFA. Peptides were separated using a nano-flow HPLC (LTQ VELOS, Thermo Finnigan, San Jose, CA, USA).

HPLC-MS analysis was carried out to identify the major components in FGE. An INNO C18 Column (150 × 4.6 mm, 5 μm, YoungJin Biochrom Co., Ltd., Seongnam, Korea) and a mixed eluent composed of (A) water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid were used as the stationary and mobile phases, respectively. The mobile phase was programmed as follow: 0 min → 5 min (10% B), 5 min → 10 min (10% B → 17% B), 10 min → 30 min (17% B → 19% B), 30 min → 40 min (19% B → 95% B), 40 min → 45 min (95% B → 95% B), 45 min → 50 min (95% B → 10% B), and 50 min → 60 min (10% B → 10% B). The flow rate and injection volume of the sample were 1.2 mL/min and 20 μL, respectively. A heated electrospray ionization probe (250 °C) with a spray voltage of 5.0 kV was used as the ionized source, and the flow rate of sheath gas (N₂) was set to 35 arb. A photodiode array (PDA) detector (Accela PDA 80 Hz; Thermo Scientific, Waltham, MA, USA) was scanned at a range of 100 to 800 nm, and MS (LTQ-Velos; Thermo Scientific, Waltham, MA, USA) was monitored at an m/z ratio of 100–1000. The data-dependent MS experiments were controlled using menu-driven software with the Xcalibur program (Thermo Electron Corporation, Waltham, MA, USA). All experiments were conducted under automatic gain-control conditions.

The LC system (Ettan MDLC, GE Healthcare, USA) was used for desalting and separation of tryptic peptides mixtures. In this system, samples were desalted on reverse phase (RP) trap columns (Zorbax 300 SB C18, Agilent Technologies, USA), and then separated on a RP column (150 μm × 100 mm, Fremont Column Technology Inc., USA). The mobile phase A was 0.1% formic acid in HPLC-grade water and the mobile phase B was 0.1% formic acid in acetonitrile. 20 μg of tryptic peptide mixtures was separated at a flow rate of 2 μL/min with a linear gradient (4–50% solution B, 50 min; 50–100% solution B, 4 min; 100% solution B, 6 min) in the columns. A mass spectrometer (LTQ Velos, Thermo Scientific, USA) was equipped with a micro-spray interface and connected to the LC setup to detecte the eluted peptides. The MS/MS spectra were set so that one full scan mass spectrum (m/z 300–1800) was followed by twenty MS/MS events of the most intense ions using dynamic exclusion (repeat count 2, repeat duration 30 seconds, exclusion duration 90 seconds).

Filter-Aided Sample Prep (FASP) was used to lyse cells, digest with trypsin, and recover the tryptic peptides [9 (link)]. Peptides were analyzed by nano-LC/MS/MS. Peptides were eluted across a 3-hour gradient on a 20 cm, self-packed C18 capillary column on an Eksigent NanoLC-2D system. Mass spectra were acquired on a Thermo LTQ Velos linear ion-trap mass spectrometer. The 5 highest intensity 2⁺ or 3⁺ ions from each MS scan were selected for collision induced dissociation (CID).

The concentration analysis of CDCA and UDCA was conducted using LC/MS/MS (Thermo Scientific LTQ-Velos). The standard CDCA and UDCA used in the analysis were purchased from Sigma-Aldrich (St. Louis, MO, USA). The extracted bile acid was dissolved in methanol and filtered using a 0.22 μm membrane filter.
The LC/MS/MS analysis conditions are shown in Table 2. The MS-MS detector was measured at 3.5 kV in negative ionization mode at 250 °C using heated electrospray ionization (H-ESI II).Table 2

Liquid chromatography–mass spectrometry (LC/MS/MS) conditions for analyzing chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA).

Table 2