0 mL/min. The isoelectric point and hydrophobic amino acid content of Hb 98–114 was calculated using the ProtParam tool, available at the ExPASy Proteomic Server (http://expasy.org/). Secondary structure prediction was performed on the NPSA server (http://npsa-pbil.ibcp.fr/) using the consensus secondary structure prediction algorithm. selleck Prior to mass spectrometry analyses, samples were desalted using C18 reverse phase tips (ZipTip, Millipore), concentrated in a SpeedVac centrifuge (Savant) and reconstituted in 5% ACN/0.2%

formic acid (FA). Molecular masses were determined by electro-spray ionization mass spectrometry (MS), using a LCQ™ Duo mass spectrometer (Thermo Scientific, USA) at a mass to charge (m/z) range from 300 to 2000 in positive mode. Equipment calibration was performed as described previously [38]. Following protein digestion with sequencing grade trypsin (Sigma, USA), samples were desalted, loaded onto a fused silica capillary column (0.1 mm × 150 mm, Polymicro Technologies, USA) in-house packed with Vydac C18 matrix (10–15 μm, 300 Å) coupled to a KRX-0401 purchase nano-HPLC system (Ultimate model, Dionex, USA). Peptides were eluted with a linear gradient from 5% to 60% ACN in 0.2% FA over 60 min. Spectral data were correlated with protein sequence databases using Bioworks Browser version 3.3 (Thermo Scientific, USA). Peptide sequences were

validated by considering a DCn ≥ 0.05 and Xcorr ≥ 1.5, 2.0 and 2.5 for singly-, doubly- and triply-charged peptides, respectively. The peptide Hb 98–114 was chemically synthesized with free amino and carboxyl terminal group and purified

as previously described [22]. It was Niclosamide structurally characterized by circular dichroism and NMR spectroscopy in phosphate buffer solutions without and with SDS micelles as membrane mimetic. CD measurements were made using a Jasco-715 spectropolarimeter. Fifty μM Hb 98–114 was prepared in 20 mM phosphate buffer and the pH was adjusted to 3, 5, 7 or 9, after which CD spectra were obtained over a range of 195–260 nm using a quartz cell of 1.0 mm path length at 25 °C. For each analysis, eight scans were accumulated and averaged. All CD spectra were corrected by subtraction of the background. The CD spectra were reported as raw ellipticity ([θ]) in mdegrees. Additionally, CD spectra were acquired in the presence of SDS micelles (25 mM SDS) added to the phosphate buffer at the different pHs described above. At pH 5, we also acquired spectra in the presence of 25 mM n-dodecyl phosphocholine (DPC) or adding increasing amounts of trifluoroethanol (TFE) from 10 to 50% (v/v). NMR experiments [16] and [35] for 1H, chemical-shift assignments and structure determination were acquired at 298 K on 500 μM Hb 98–114 dissolved in 5 mM phosphate buffer, 25 mM NaCl at pH 5.6 using 10% D2O for the deuterium lock signal in the presence or absence of 100 mM SDS-d25 (98% deuterium, Cambridge Isotopes Inc.).