In-solution digestion and sample preparation were performed as described previously (Wiśniewski et al., 2009 (link)). Peptides were separated using an UHPLC Dionex UltiMate® 3000 (Thermo Fisher Scientific, USA) instrument. Tryptic digests were separated by reversed-phase chromatography. Fractions were reconstituted in solvent A (water/acetonitrile, 98:2 v/v; 0.1% formic acid). The UHPLC was equipped with a Acclaim PepMap 100 trap column (100 μm × 2 cm, nanoViper C18, 5 μm, 100 Å) to trap the sample, which was subsequently washed with 98% solvent A for 6 min at a flow rate of 6 μL/min, and then continuously separated on a Acclaim PepMap 100 capillary column (75 μm × 15 cm, nanoViper C18, 3 μm, 100 Å) at a flow rate of 400 nL/min. The LC analytical gradient was run at 2% to 35% solvent B over 90 min, then 35% to 95% over 10 min, followed by 90% solvent B for 5 min, and finally 5% solvent B for 15 min. Resulting peptides were electro-sprayed through a coated silica tip (PicoTip emitter, New Objective, USA) at an ion spray voltage of 2,000 eV.
The UHPLC was coupled with a heated electrospray ionization source (HESI-II) to the quadrupole-based mass spectrometer QExactiveTM Orbitrap High Resolution Mass Spectrometer (Thermo Fisher Scientific, USA). The MS spectra were acquired at a resolution of 70,000 (200 m/z) in a mass range of 350-1,800 m/z. A maximum injection time was set to 100 ms for ion accumulation. Eluted samples were used for MS/MS events, measured in a data-dependent mode for the 10 most abundant peaks (Top10 method), in the high mass accuracy Orbitrap after ion activation/dissociation with Higher Energy C-trap Dissociation (HCD) at 27 collision energy in a 100-1650 m/z mass range.
The UHPLC was coupled with a heated electrospray ionization source (HESI-II) to the quadrupole-based mass spectrometer QExactiveTM Orbitrap High Resolution Mass Spectrometer (Thermo Fisher Scientific, USA). The MS spectra were acquired at a resolution of 70,000 (200 m/z) in a mass range of 350-1,800 m/z. A maximum injection time was set to 100 ms for ion accumulation. Eluted samples were used for MS/MS events, measured in a data-dependent mode for the 10 most abundant peaks (Top10 method), in the high mass accuracy Orbitrap after ion activation/dissociation with Higher Energy C-trap Dissociation (HCD) at 27 collision energy in a 100-1650 m/z mass range.
