Sciex 5800

In vitro alanylation and de-alanylation reactions were carried out as described before^{68 (link)}. In vitro alanylation was carried out in a 30-μl reaction mix containing 50 mM HEPES (pH 7.5), 25 mM KCl, 2 mM MgCl₂, 5 mM alanine, 4 mM ATP, 10 nM AARS1, and 0.05 mg/ml synthetic substrate peptide. The final pH of each reaction mix was adjusted to 7.5 before adding AARS1. The reaction was allowed for 3 h at 37 °C. The peptide was desalted by passing through a C18 ZipTip (Millipore) and was subjected to analyzation by a MALDI-TOF/TOF mass spectrometer (SCIEX-5800).
In vitro de-alanylation reactions were carried out in a 30-μl reaction mix containing 50 mM HEPES (pH 7.5), 6 mM MgCl₂, 1 mM DTT, 1 mM NAD+, 0.05 mg/ml synthetic peptide, 1 mg/ml SIRT6 and 1 mM PMSF. The reaction was allowed for 4 h at 37 °C. The peptide was desalted by passing through a C18 ZipTip (Millipore) and was subjected to analyzation by a MALDI-TOF/TOF mass spectrometer (SCIEX-5800).

The MALDI-TOF–MS (AB SCIEX 5800) was used to record the mass spectrum of the purified IMOs sample. An equivalent volume of matrix solution (0.1 M 2,5-dihydroxybenzoic acid and 0.03 M 1-hydroxyisoquinoline in aq. 50 percent ACN) was mixed with an aqueous sample. The sample mixture was loaded on a MALDI target plate and dried^{41 (link)}. A mass spectrum was generated by using the spectra from 200 laser pulses^{42 (link)}.

The position and molecular weight of the bands in the two gels were compared and grayscale analysis was carried out with Image J software (http://imagej.nih.gov/ij/plugins/). The samples with statistically significant differences (p < 0.05) were selected for MS mass spectrometry analysis. SDS-PAGE electrophoresis was performed with 1.5 mg protein sample according to the above western blot steps. The gel was stained with Coomassie brilliant blue solution (containing 0.12% Coomassie Brilliant Blue G-250, 20% ethanol, 10% phosphoric acid, 10% ammonium sulfate). After stainning, manually remove the band of protein ladder from the gel. Each gel was digested with trypsin (Promega Corp, WI, USA) overnight at 37 ℃, for MALDI-TOF–MS mass spectrometry.
MALDI-TOF/TOF mass spectrometer (AB SCIEX 5800, USA) was used for protein identification, 0.6 μL of protein sample was treated with 1 μL of 10 mg/ml α-cyano-4-hydroxycinnamic acid (CHCA) at 0.1% TFA, 50% acetonitrile (ACN), directly crystallized on the target and dried at room temperature for detection. The equipment is set in linear mode, ion acceleration voltage 20 kV, N2 laser wavelength 337 mm, pulse width 3 ns, ion delay extraction 500 ns, vacuum 5 × 10⁵ Pa, the spectrum is externally calibrated.