Twenty micrograms of the HBV core 131–162, 32-mer polypeptide and 3 μg of purified proteasome complexes were incubated in 300 μl TEAD buffer at 37 °C for the indicated time (37 μl per time point). The reaction was terminated by the addition of trifluoroacetic acid. The digested products were separated by reversed-phase chromatography on a 1 mm RP column (Beta Basic-18, 100 × 1 mm, 3 μm, 150 Å, Thermo Scientific). Peptides were detected online with electrospray ionization–mass spectrometry (DECA XP MAX iontrap instrument; Thermo Scientific). The kinetics of the identified peaks in time-dependent processing experiments (signal intensity versus time of digestion) was analyzed using the LCQuan software version 2.5 (Thermo Scientific).
Beta basic 18
Manufactured by Thermo Fisher Scientific
The Beta Basic-18 is a laboratory equipment that serves as a basic water bath. It is designed to provide a controlled temperature environment for various applications in the laboratory setting.
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4 protocols using beta basic 18
1
Proteasome-Mediated Degradation of HBV Core Peptide
2
Multimodal Analytical Characterization Protocol
3
Determination of Ascorbic Acid and β-Carotene using HPLC
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Ascorbic acid was determined using HPLC methods of AOAC [29 (link)] with slight modification. The separation was performed on a Thermo Electron Co., column BetaBasic-18 (150 mm × 4.6 mm, 5 µm). The isocratic mobile phase consisted of 0.1% o-phosphoric acid in water.
β-carotene was determined using HPLC normalised methods of EN-12823-2:2000 [30 ] with slight modification. The separation was performed on a Kinetex C18 (Phenomenex) column (150 mm × 4.6 mm, 2.6 µm).
β-carotene was determined using HPLC normalised methods of EN-12823-2:2000 [30 ] with slight modification. The separation was performed on a Kinetex C18 (Phenomenex) column (150 mm × 4.6 mm, 2.6 µm).
4
Quantification of Lidocaine and MEGX
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Stock solutions (1 mg/mL) of lidocaine hydrochloride monohydrate (Dr Ehrenstorfer), MEGX (Sigma Aldrich) and lidocaine HCl d10 (internal standard, CDN Isotopes) were prepared in methanol and stored at -20 °C.
Serum samples (100 µL) were added to 100 µL of internal standard and 800 µL of acetonitrile, vortexed and then centrifuged at 18,000 g for 5 min. Organic layers (100 µL) were diluted with 400 µL of acetonitrile and 10 µL were injected in the LCsystem. Quantitative analyses were conducted by means of a triple quadrupole mass spectrometer (TSQ Quantum Ultra, Thermo Fisher) equipped with a LC-system (Finnigan Surveyor LC pump, Thermo Fisher) via electrospray ionization (ESI) interface. The chromatographic analytical column was a BetaBasic-18 (150 mm 2.1 mm, 5 µm, Thermo Electron Corporation). The separation was performed in gradient mode, using as mobile phases water and acetonitrile, both containing 0.1% (V/V) formic acid; the flow rate was 0.3 mL/min. The ESI source operated in positive ion mode. The parameters were as follows: spray voltage 3.5 kV and capillary temperature 270 °C. The concentration of lidocaine or MEGX was determined by the internal standard method.
Serum samples (100 µL) were added to 100 µL of internal standard and 800 µL of acetonitrile, vortexed and then centrifuged at 18,000 g for 5 min. Organic layers (100 µL) were diluted with 400 µL of acetonitrile and 10 µL were injected in the LCsystem. Quantitative analyses were conducted by means of a triple quadrupole mass spectrometer (TSQ Quantum Ultra, Thermo Fisher) equipped with a LC-system (Finnigan Surveyor LC pump, Thermo Fisher) via electrospray ionization (ESI) interface. The chromatographic analytical column was a BetaBasic-18 (150 mm 2.1 mm, 5 µm, Thermo Electron Corporation). The separation was performed in gradient mode, using as mobile phases water and acetonitrile, both containing 0.1% (V/V) formic acid; the flow rate was 0.3 mL/min. The ESI source operated in positive ion mode. The parameters were as follows: spray voltage 3.5 kV and capillary temperature 270 °C. The concentration of lidocaine or MEGX was determined by the internal standard method.
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