Hybrid quadrupole tof lc ms ms mass spectrometer

Two hundred mg of each faecal sample and 1 ml of ethyl acetate were mixed by vortexing. The mixtures were incubated on ice for 30 min and were then centrifuged at 12 000 rpm at 4°C for 10 min. Then, 800 µl of supernatant was extracted and concentrated to dryness in vacuum at room temperature. The residue was reconstituted with 200 μl of methanol and filtered using a 0.22‐μm filter membrane. 10 μl of the resulting solution was aspirated for analysis.
The analysis was performed using a Shimadzu LC30A coupled with a Hybrid Quadrupole‐TOF LC/MS/MS Mass Spectrometer (AB SCIEX, Framingham, MA, USA) in the positive mode. A waters ACQUITY UPLC Xbridge C18 Column (2.5 µm, 2.1 mm × 150 mm) was used with a flow rate at 0.2 ml min^‐1 and a column temperature of 55°C. The eluents were (A) 0.1% FA in water and (B) acetonitrile. The gradient was set as follows: 5% B at 0 min, 50% B at 7 min, 100% B at 10 min, 100% B at 15 min, 5% B at 16 min and 5% B at 18 min.

Accurate molecular mass and amino acid sequence of the active peptide was determined by Hybrid Quadrupole TOF-LC/MS/MS mass spectrometer (AB Sciex Instruments, CA, USA) coupled with ESI source. Generally, the peptide was infused into electrospray source following dissolve in acetonitrile/water (1:1, v/v), and molecular mass was determined by charged (M + H)⁺ state in the mass spectrum. Following molecular mass determination and sequence information was obtained by BioLynx analysis system.

Protein bands obtained from the binding assay were subjected to in-gel digestion with trypsin^{80 (link),81 (link)}. The digested peptide solution was desalted and concentrated, and was eluted using a homemade C18 nano-column (100–300 nl with trypsin of POROS reverse-phase R2 material (20–30 μm in bead size, PerSeptive Biosystems, Foster City, CA)) and 1.5 ul of 50% MeOH, 49% H₂O, and 1% HCO₂H. qTOF-MS of the eluted peptides was performed using a Hybrid Quadrupole-TOF LC/MS/MS Mass Spectrometer (AB Sciex Instruments, Framingham, MA) equipped with an electrospray ionization (ESI) source. The quadrupole analyzer was used to select precursor ions for fragmentation in the hexapole collision cell. The produced ions were analyzed using an orthogonal TOF analyzer and fitted with a reflector, a micro-channel plate detector, and a time-to-digital converter.