Orbitrap velos elite mass spectrometer

Sample injections of 11 μL containing 600 ng digested peptide was loaded with blank runs intervening between each sample. The cercarial heads and tails were run in triplicate. The Orbitrap Velos Elite mass spectrometer (Thermo Electron, San Jose, CA) equipped with the Waters nanoACQUITY LC system (Waters, Taunton, MA) was used for acquisition. Peptides were desalted in a trap column (180 μm × 20 mm, packed with C18 Symmetry, 5 μm, 100 Å, Waters, Taunton, MA) and subsequently resolved in a reversed-phase column (75 μm × 250 mm nano column, packed with C18 BEH130, 1.7 μm, 130 Å (Waters, Taunton, MA). Liquid chromatography was carried out at ambient temperature at a flow rate of 300 nL/min using a gradient mixture of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The gradient employed ranged from 4 to 44% solvent B over 210 min. Peptides eluting from the capillary tip were introduced into the nanospray mode with a capillary voltage of 2.4 kV. A full scan was obtained for eluted peptides in the range of 380–1800 atomic mass units, followed by twenty-five data-dependent MS/MS scans. MS/MS spectra were generated by collision-induced dissociation of the peptide ions at a normalized collision energy of 35% to create a series of b- and y-ions as major fragments. A one-hour wash was included between each sample.

For each sample, 400ng of AM and MN peptide digests were loaded on a column in a 14μL injection. Samples were randomized and blanks were added between samples. Resulting data was acquired on an Orbitrap Velos Elite mass spectrometer (Thermo Electron, San Jose, CA) equipped with a Waters nanoAcquity LC system (Waters, Taunton, MA). Peptides were desalted in a trap column (180 μm × 20 mm, packed with C18 Symmetry, 5μm, 100Å, Waters, Taunton, MA) and subsequently resolved in a reversed phase column (75μm x 250 mm nano column, packed with C18 BEH130, 1.7μm, 130Å (Waters, Taunton, MA)). Liquid chromatography was carried out at ambient temperature at a flow rate of 300 nL/min using a gradient mixture of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The gradient employed ranged from 4 to 44% solvent B over 210 min. Peptides eluting from the capillary tip were introduced into the nanospray mode with a capillary voltage of 2.4 kV. A full scan was obtained for eluted peptides in the range of 380–1800 atomic mass units followed by twenty-five data dependent MS/MS scans. MS/MS spectra were generated by collision-induced dissociation of the peptide ions at normalized collision energy of 35% to generate a series of b- and y-ions as major fragments. A one-hour wash was included between each sample.