Screening of Syk Affinitive Components from PLE

After Dynabeads MyOne Carboxylic Acid (Invitrogen, California, USA) were activated with 50 μL EDC (50 mg/mL dissolved in MES) and 50 μL NHS (50 mg/mL dissolved in MES), 60 μL Syk (1 mg/mL dissolved in MES) and 40 μL MES were added to beads and allowed to incubate for 16 h at 4°C. The beads (Syk targeted) were then washed 3 times with MES, vortexed and incubated with 600 μL PLE (20 mg/mL) for 2 h at room temperature (RT). Then, the beads were sampled on a MS separation column with a MiniMACS^TM separator (Miltenyi Biotec, Berlin, Germany) and washed with appropriate amount of water 3 times. Individual eluents were collected upon the addition of 600 μL of 10%, 50% and 100% methanol. All the eluents were then concentrated to 600 μL volume. Uncoated beads were used as control (Blank).

The Syk affinitive components were characterized and identified by LC-MS/MS (QSTAR Elite LC-MS/MS System, Massachusetts, AB SCIEX, USA). Electrospray ionization was operated in a negative ion mode. EC-C18 column (4.6×100 mm, 2.7 μm, Agilent, California, USA) was used in reversed-phase mode, and the column temperature throughout the analysis was 30°C. The injection volume was 20 μL. The mobile phase (at a flow rate of 1 mL/min) consisted of water with 0.1% formic acid (A) and acetonitrile (B). The gradient elution was set as follows: 0–15 min, 90–80% A; 15–30 min, 80–60% A; 30–45 min, 60–40% A; 45–60 min, 40–0% A. Fragments were detected within the m/z range 100–1000. The standards of RosS, Vic-2 and RosA were used to confirm identity of compounds analyzed by LC-MS/MS.