4000 qtrap lc ms ms

The HPLC system consisted of two LC-20AD pumps, a DGU-20 A3 degasser, an SIL-20AC autosampler and a CTO-20A column oven (Shimadzu, Tokyo, Japan). All targeted compounds were separated on a reverse phase Poroshell 120 SB C₁₈ column (100 mm × 3.0 mm, 2.7 μm, Agilent Technologies, Santa Clara, CA, USA), which was eluted with a gradient mobile phase consisting of (A) water (containing 0.1% formic acid and 5 mM ammonium formate) and (B) acetonitrile. A two-step gradient elution program was utilized as follows: 0.0 min 20% B, 8.0 min 25% B, 15.0 min 60% B, 15.1 min 20% B, 18.0 min 20% B. The flow rate was set at 0.4 mL/min. The column effluent was monitored using a 4000 QTRAP^® LC-MS/MS (AB Sciex, Toronto, ON, Canada).
The ESI source was operated in positive mode with the curtain, nebulizer and turbo-gas (all nitrogen) set at 20, 50 and 55 psi, respectively. The turbo-gas temperature was 500 °C and the ion spray needle voltage was 5500 V. The compound-dependent instrumental parameters of two individual precursor-to-product ion transitions specific for each analyte including precursor ion, two product ions, declustering potential (DP), entrance potential (EP), collision energy (CE) and collision cell exit potentials (CXP) were optimized and are listed in Table 3. The dwell time was 200 ms for each MRM transition.

Peptides 1, 2, and 3 have been synthesized as previously reported [44] (link), [49] (link).
Detailed information on mass spectrometric measurements is provided in the supporting information (Method S1). Briefly, the 4000 QTRAP LC/MS/MS and the 6500 QTRAP LC/MS/MS systems (AB Sciex Germany GmbH, Table S1) were used. For MS full scans experiments signal intensity was adjusted to achieve 1e6 counts per second (cps) maximal signal intensity at 10 µL per minute infusion from a syringe pump. Peptide stock solutions have been prepared by dissolving 0.5 mg of the respective peptide in 1 mL of a mixture of 20% methanol (Fluka Analytical, LC-MS CHROMASOLV) and 80% water (Merck KGaA, LC-MS LiChrosolv) with 0.2% formic acid (Fluka Analytical, analytic additive).
For MS² experiments both declustering potential (DP) and collision energy (CE) have been optimized via ramping. Regarding MS³ measurements, resonance excitation energy (AF2) was additionally optimized (using ‘ramping’ functionality of Analyst software). All spectra were accumulated to ensure distinct differentiation between signals and noise.

Serum samples (100 µL) and prechilled methanol (400 µL) were mixed by well vortexed. Cecal contents, liver, and adipose tissues (≈10 mg) were individually grounded with liquid nitrogen and the homogenate was resuspended with prechilled 80% methanol and 0.1% formic acid by well vortexed. The samples were incubated on ice for 5 min and then were centrifuged at 15 000 rpm, 4 °C for 5 min. The supernatants were diluted to final concentration containing 53% methanol by LC‐MS/MS grade water. The samples were subsequently transferred to a fresh Eppendorf tube and then were centrifuged at 15 000 g, 4 °C for 10 min. The supernatant was analyzed for tryptophan, I3AA, and other indole derivatives using 4000 Q TRAP LC‐MS/MS (AB Sciex) analysis according to a previously described method (Table S5, Supporting Information).^[33 (link)
^] Specifically, L‐tryptophan (T0254, Sigma), I3AA (45533, Sigma), indole (442619, Sigma), 3‐indoleacrylic acid (I2273, Sigma), indole‐3‐carboxaldehyde (129445, Sigma), and indole‐3‐lactic acid (I157602, Aladdin) were used as standards in the current study. The absolute concentration of metabolites was calculated according to the standard curves, which were created using six appropriate serial dilutions of the corresponding standards.