Compass hystar software package

Most of PETases can break the ester bond of BHET to produce TPA and ethylene glycol (EG) with MHET as intermediate product^{17 (link),18 ,38 (link)}. Therefore, BHET is often selected as the representative substance to study the activity of PETase^{17 (link),39 (link)}. In this study, BHET and in vitro expressed PETase candidates were incubated in phosphate-buffered saline (PBS, Carl Roth, Karlsruhe, Germany) at indicated temperatures for 4 days. The yields of TPA and MHET were quantified with UltiMate 3000 UHPLC system from Thermo Scientific with a Triart C18 column (YMC Europe GmbH, Dinslaken, Germany) and a VWD-3400 detector (Thermo Scientific)¹⁹ . The data were analyzed with Compass HyStar software package from Bruker (Billerica, MA, USA).

The UHPLC–MS/MS
analysis
was performed in a Maxis II ETD Q-TOF mass spectrometer (Bruker Daltonics,
Germany) using an electrospray ionization (ESI) source with either
an Elute UHPLC (Bruker Daltonics, Germany) or a 1260 Infinity II Binary
Pump (Agilent Technologies, USA) system. The separation was performed
on an Acquity UPLC HSS T3 column (1.8 μm, 100 × 2.1 mm)
from Waters Corporation. Milli-Q water with 0.1% formic acid was used
as mobile phase A, and LC–MS grade methanol with 0.1% formic
acid was used as mobile phase B. The column temperature was kept at
40 °C, and the autosampler temperature was kept at 4 °C.
The flow rate was set to 0.22 mL/min with an injection volume of 5
μL. The gradient used was as follows: 0–2 min, 0% B;
2–15 min, 0–100% B; 15–16 min, 100% B; 16–17
min, 100–0% B; 17–23 min, 0% B. The system was controlled
using the Compass HyStar software package from Bruker (Bruker Daltonics,
Germany). High-resolution mass spectra were acquired in negative mode
at a mass range of m/z 50–1200.
Data acquisition was performed in AutoMSMS mode (data-dependent acquisition,
DDA) with a cycle time of 0.5 s and a ramped collision energy from
20 to 50 eV. A solution of sodium formate [10 mM in a mixture of 2-propanol/water
(1/1, v/v)] was used for internal calibration at the beginning of
each run, in a segment between 0.10 and 0.31 min.

The analysis of TPA concentrations in supernatants of enzymatic incubations with PET was performed using an UltiMate 3000 UHPLC system from Thermo Scientific (Waltham, MA, USA). The system was fitted with a Triart C18 column (YMC Europe GmbH, Dinslaken, Germany) having a dimension of 100 × 2.0 mm with 1.9 μm particle diameter. Isocratic elution was carried out in a 20:80 (vol/vol) acetonitrile and water (acidified with 0.1% vol/vol trifluoroacetic acid) mobile phase at 0.4 mL min⁻¹. Samples were prepared from 50 μL of incubation supernatant mixed with 200 μL acetonitrile (acidified with 1% vol. trifluoroacetic acid), followed by centrifugation at 10,000 × g for 3 min. 200 μL of the supernatant was transferred to 600 μL water. 15 μL of sample were injected per measurement. Detection TPA was carried out at 254 nm with a VWD-3400 detector from Thermo Scientific (Waltham, MA, USA). The quantification of peaks was performed with the data analysis software supplied with the Compass HyStar software package from Bruker (Billerica, MA, USA).