Acetic anhydride d6

Chemical acetylation of Rubisco was performed according to the method of [39 (link)]. Rubisco samples (100 µg) were resuspended in 100 µl of 8 M urea, 200 mM triethylammonium bicarbonate followed by the addition of 2 µl of 1 M dithiothreitol (DTT) and incubation at 37°C for 30 min. Twenty-two microlitres of 200 mM IAA was then added, followed by incubation at room temperature for 1 h. Three rounds of chemical acetylation were then performed by adding 6 µl of acetic anhydride-d₆ (10.57 M; Sigma) and incubating at 4°C for 20 min, followed by neutralisation to approximately pH 7.5 with 7.5 M NaOH. Ten microlitres of 50% hydroxylamine solution was then added to revert O-acetylation side reactions. Samples were then digested with 1 µg of trypsin overnight at 37°C. Samples were then subject to solid-phase extraction using C18 MacroSpin Column (The Nest Group, Inc.) according to the manufacturer's instructions, eluted in 70% acetonitrile, 0.1% formic acid and dried down in a vacuum desiccator.
Orbitrap MS/MS analysis of Rubisco samples was then performed as above. MaxQuant analysis of MS/MS data was performed as above except that Lys-acetylation-D₃ (C₂D₃O) was added as a variable modification and missed cleavages were set at 5.

Escherichia coli strain MG1655 was transformed with either pBAD33-ItaT or empty pBAD33 and cultured overnight. The overnight cultures were diluted to an OD₆₆₀ of 0.03 into fresh liquid LB (3 ml) containing 50 μg/ml chloramphenicol, and the cultures were continued at 37°C until the A₆₆₀ reached 0.2. At this point, 0.02% (w/v) arabinose was added. At 15 min after inoculation, the cells were harvested, and suspended in buffer containing 50 mM NaOAc, pH 5.0, 0.5 mM EDTA and 0.2 M NaCl. The RNA was extracted by phenol saturated with 300 mM NaOAc, pH 5.2, followed by isopropyl alcohol precipitation. The RNA was dissolved in 250 μl of cold 200 mM NaOAc, pH 5.0, and acetylated by adding acetic anhydride-D₆ (Sigma Aldrich, Japan) as described (28 (link)). Afterwards, the RNA was ethanol precipitated and rinsed with 70% cold ethanol. The RNA was dissolved in cold buffer containing 50 mM NaOAc, pH 5.0, 0.5 mM EDTA and 0.2 M NaCl, and loaded onto 100 μl of Q-Sepharose FF (GE Healthcare, Japan). The resin was washed with buffer containing 50 mM NaOAc, pH 5.0, 0.5 mM EDTA and 0.2 M NaCl. The tRNA was eluted with buffer containing 50 mM NaOAc, pH 5.0, 0.5 mM EDTA and 0.6 M NaCl, ethanol precipitated and rinsed with 70% ethanol. The pellet was dissolved in 2 mM NaOAc, pH 5.0.