α cyano 4 hydroxycinnamic acid

The purified protein was run on a 12% SDS-PAGE gel and the lane containing the target protein was removed and cut into pieces of about 1 mm³. After washing three times with sterile water, the gel pieces were destained by three sequential 15-min treatments with 100 μL of 25 mM ammonium bicarbonate (Fluka) in 50% (v/v) acetonitrile (Fisher) at pH 8.0, and the pieces where then washed once more with sterile water. The gel slices were dehydrated with 30 μL 100% acetonitrile and completely dried with a Speed-Vac at room temperature. The samples were digested with 8 μL of 0.1 mg/mL trypsin (Promega) overnight at 37 °C. Then, 0.3 μL of the digested protein was mixed with 0.3 μL of matrix solution (5 mg/mL α-cyano-4-hydroxycinnamic-acid (Fluka) in 50% (v/v) acetonitrile (Fisher) and 0.1% (w/v) trifluoroacetic acid (DIMA)) and spotted onto a sample plate for matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry analysis. Positive ion reflection mode was selected and the results were analyzed using 4000 Series software. The target protein was identified by comparing the peptide sequences against homologous proteins in the SwissProt databases using the following parameters: precursor tolerance, ±0.2 Da; MS/MS tolerance, ±0.8 Da; missed cleavages, 1.