For protein purification, further additional steps of chromatography were necessary, using a Bio Basic C8 column (4.6 mm × 250 mm, 5 μm, Thermo, USA) with optimized Z-VAD-FMK solubility dmso gradients. The HPLC column eluates were monitored by their absorbance at 214 nm. SDS-PAGE

was carried out according to Laemmli (1970). Proteins (10 μg) from the mucus of P. cf. henlei were analyzed by SDS-PAGE 4–20% acrylamide gradient under reducing conditions. Prior to electrophoresis, the samples were mixed 1:1 (v/v) with sample buffer. The gels were stained with the Silver method. The fractions were analyzed by electrospray, with direct injection in an LC–MS Surveyor MSQ Plus (Thermo Electron, USA) under positive ionization mode. The needle and cone potential were set to 3.1 kV and 40 V, respectively. The aqueous sample solutions (10 μL) were directly injected at a 50 μL/min constant flow rate of acetonitrile H2O/0.1% formic acid (1:1). External calibration was performed with NaI (Sigma) over m/z 100–2000. Protein band was excised and in-gel trypsin digestion was performed according to Hellman et al. (1995). Nanospray MS/MS analysis was performed on tryptic digests of SDS-Page band of purified PcfHb using Q-Tof mass spectrometry (Q-TOF Ultima API Waters/Micromass, Manchester, United Kingdom). An aliquot (5 μL) of the

resulting peptide mixture were injected into Symmetry C18 trapping column (5 μm particles, 180 μm i.d. × 20 mm, Waters, USA) to desalt the peptide mixture.

5-Fluoracil cost The nano UPLC (Waters) conditions were 0.1% O-methylated flavonoid formic acid in water (solvent A) and acetonitrile with 0.1% formic acid as solvent B. The separations were performed at a flow rate of 0.6 μl/min using a 0–80% gradient of solvent B over 45 min. The LC system was coupled to a nano ESI source of the Q-ToF instrument using a BEH130C18 column (75 μm × 100 mm, 1.7 μm particles; Waters, MA, USA). Typical conditions were a capillary voltage of 3.1 kV, a cone voltage of 50 V, and source temperature of 70 °C. Data dependent acquisition (parent ions with 2, 3 and 4 charges) were automatically recognized by the charge state recognition software MassLynx 4.1 (Waters, USA). The peptide ions were detected by scanning from m/z 200 to m/z 2000 at a rate of 1 scan/s, and were subjected to collision-induced dissociation with argon in the 13–55 eV collision energy range. Product ions from MS/MS experiments were detected by scanning from m/z 50 to m/z 2000 at a rate of 1 scan/s. External calibration was performed using phosphoric acid (Merck, Darmstadt, Germany). All MS/MS spectra were acquired using MassLynx 4.0 software (Waters), deisotoped and converted (by Waters ProteinLynx Global server 1.0 software) and searched using a licensed copy of the Mascot Server 2.2 program (Matrix Science, London, UK). In each instance, the search was carried out against the non-redundant protein database at the National Center for Biotechnology Information bank (NCBI www.ncbi.nlm.nih.