Waters lc system

A previous report was used as a reference (14 (link)). Ten microgram of total protein was used in the PDC assay in a volume of 100 μl. After incubating the mixture at 37°C for 10 min, the reaction was stopped by adding 100 μl of 100 mM glycine–HCl pH 2.5. The mixture was then filtered with an Amicon Ultra 3K device by centrifugation for 15 min at 15 000 rpm, 4°C. The product, acetyl-CoA, was measured by LC/MS/MS. LC/MS/MS analysis was carried out using a Quattro Ultima Pt triple stage quadrupole mass spectrometer (Waters) equipped with a Waters LC system. The LC separation was according to a previous report (15 (link)). Multi-reaction monitoring was performed in negative ion mode using nitrogen as the nebulizing gas. Experimental conditions were as follows: ion source temperature, 130°C; desolvation temperature, 380°C; capillary voltage, 2.4 kV; cone voltage, 35 V; collision energy, 15 eV; desolvation gas flow rate, 700 L/h; cone gas flow rate, 35 L/h; collision gas, argon. The MRM transition for acetyl-CoA was m/z 808.0 → 407.7. The amount of acetyl-CoA was quantified by calculating the peak area ratio of acetyl-CoA. The kinetics parameters (K_m and V_max) were fitted to a Hanes–Woolf plot.

RAB33B proteins with a defined nucleotide-binding state were obtained by nucleotide exchange. Ethylenediaminetetraacetic acid (EDTA) (Sigma, E9884) at a final concentration of 5 mM (2.5-fold higher than the concentration of MgCl₂) was added into the RAB33B protein. Subsequently, 20-fold excess of GppNHp (Jena Bioscience, NU-899), GTP or GDP was added and the solution was incubated at room temperature for 2 h. The protein solution was subjected to buffer exchange [20 mM HEPES (Carl Roth, 6763.3), pH 7.2, 25 mM NaCl (Carl Roth, P029.2), 2 mM DTE (Sigma, D8255), 2 mM MgCl₂ (Carl Roth, HN03.1) and 10 µM respective nucleotide)] using a NAP5 column (GE Healthcare, 17–0853-01) to remove excess EDTA (Sigma, E9884) and nucleotide. The state of the nucleotide binding was determined by high-performance liquid chromatography using ProntoSIL 120-5-C18-AQ 5 µm (250 x 4.6 mm) column (Bischoff) eluted with buffer [50 mM potassium phosphate (prepared from dipotassium hydrogen phosphate, Sigma, P3786), pH 6.6, 10 mM tetrabutylammonium bromide (Sigma, 193119), 8% acetonitrile (Sigma, 34851)] in a Waters LC system (Waters Corp.).