Esi ion max source

The HPLC-separation of red wines conducted as described above afforded three fractions for each wine. The first fraction was collected from 15 to 25 min (fraction 1), the second one from 25 to 30 min (fraction 2) and the third fraction (fraction 3) from 30 min through the end of the chromatographic run (45 min). Each collected fraction was dried, solubilized in methanol and analyzed by HR-ESIMS in continuous flow injection in the positive ion mode. HR ESIMS experiments were performed on an Agilent 1260 Infinity II HPLC quaternary system coupled to a linear ion trap LTQ Orbitrap XL hybrid Fourier transform MS (FTMS) instrument equipped with an ESI ION MAX source (Thermo-Fisher). The following source settings were used (mass range m/z 100–2000): spray voltage 4.5 kV, capillary temperature 300 °C, capillary voltage 15 V, sheath gas 20 and auxiliary gas 21 (arbitrary units), tube lens voltage 140 V, and 25% collision energy. Calculation of elemental formulae was conducted by using Xcalibur software v 2.0.7 with a mass tolerance constrain of 5 ppm.

100 μg of each fusion protein was reduced in denaturing conditions by incubation in 50 mM tris (2-carboxyethyl)phosphine (TCEP) and 4 M guanidinium chloride for 1 h at room temperature. The samples were then desalted using C18 Micro Spin Columns (Harvard Apparatus). The eluate from C18 purification was adjusted to 48% CH₃CN/0.2% HCOOH and then directly injected into a Q Exactive mass spectrometer (Thermo Scientific) equipped with an ESI Ion Max source (Thermo Fisher). Source parameters were set as polarity positive ion mode; resolution (FWHM at 200 m/z) 70,000; microscan 10; S-lens RF level 55; spray voltage 3.5 kV; and scan range 100–1500 m/z. By applying an in-source CID offset voltage of 80 eV fragmentation of L19-IFNg variants was induced. The resulting fragmented spectra were screened for the presence of y ions, which contain C-terminal sequence information and they were manually annotated allowing a mass error of 10 ppm.