Ultimate 3000 rslc system

For TMT labeling, the peptide samples of the 4 groups were separately labeled using 4 labeling reagents of the 6-plex label sets (Thermo Scientific, USA). Each sample containing 30 μg protein was resuspended in 50 μl TEAB buffer containing 60% acetonitrile (ACN) and mixed with 20 μl of the corresponding TMT reagent for 2 h at 26 °C. The labeling reactions were then quenched by adding 5% hydroxylamine for an additional 30 min incubation. Four labeled peptide samples were mixed in equal amounts and dried in a centrifugal evaporator.
The mixed TMT-labeled peptides were resuspended in 110 μl of mobile phase A (20 mM ammonium formate buffer, 3% ACN, pH 10.0) and separated using a 1.7 μm × 2.1 mm × 100 mm BEH C18 column (Waters, USA) in a Dionex Ultimate 3000 RSLC system with a 60 min gradient starting from 4% mobile phase B (ACN) to 64% B at a flow rate of 0.25 ml·min^-1. Eluted fractions were pooled into 10 peptide samples over the gradient at 1 min intervals and were dried to completion. The peptide samples were reconstituted in 0.1% formic acid for the subsequent LC-MS/MS analysis.

Analysis of lactic acid, sugars and other fermentation products of B. coagulans which may occur such as ethanol and acetic acid was performed using a Waters e2695 HPLC system (Milford USA) equipped with Waters RI2414 and Waters UV/Vis 2489 (measuring at 210 nm) detectors. The column used was a Shodex RS pak KC-811 ion exchange column (length 300 mm, I.D. 8 mm), controlled at 65 °C. As eluent, 3 mM H₂SO₄ in milli-Q water was used. The flow used was 1 mL/min. Samples obtained during fermentation were de-frozen prior to analysis. Two hundred fifty microlitres of this sample was mixed with 250 μL of internal standard, containing 0.25 g/L phthalic acid and 500 μL of milli-Q water. Samples were filtered using 0.2 μm Spartan filters, and supernatants were measured using HPLC.
To determine furan concentrations, UPLC-MS/MS measurements were performed using a Dionex Ultimate 3000 RSLC system, equipped with a Waters Acquity BEH C18 RP column, in combination with a Thermo Scientific^TM LCQ Fleet Ion Trap Mass Spectrometer, as previously described (van der Pol et al. 2015 (link)).

All analytical LC-MS measurements were performed on a Dionex Ultimate 3000 RSLC system using a BEH C₁₈, 100- by 2.1 mm, 1.7-μm particle diameter (dp) column (Waters, Germany), coupled to a maXis 4G high-resolution time of flight (HR-ToF) mass spectrometer (Bruker Daltonics, Germany) using an Apollo electrospray ionization (ESI) source. UV spectra were recorded by a diode array detector (DAD) in the range of 200 to 600 nm. The LC flow was split to 75 μl/min before entering the mass spectrometer.