2.3. Liquid chromatography and mass spectrometry

Liquid chromatography of tryptic peptides was performed using the Ultimate 3000 RSLCnano system. Peptides in a volume of 2 µL (totally 1 µg of peptides) were loaded on an enrichment column Acclaim µ-Precolumn (0.5 mm × 3 mm, 5 µm) (Thermo Scientific, USA) in a mobile phase comprised of 0.1% formic acid and 0.03% acetic acid at a flow rate 15 µL/min. After 3 min of loading, peptides were departure from the enrichment column and separated on the Acclaim Pepmap® C18 column (75 µm × 100 mm, 2 µm) (Thermo Scientific) at a flow rate 0.3 µL/min in a linear gradient of the mobile phase A (water with 0.1% formic acid and 0.03% acetic acid) and the mobile phase B (acetonitrile with 0.1% formic acid and 0.03% acetic acid). The eluting scheme started from 2% to 37% of the mobile phase B during 45 min, then column washing in 90% of mobile phase B for the next 8 min, followed by the column equilibration under the starting condition for the next 15 min.

Detection of peptides was performed using an ultra-high resolution Orbitrap Fusion (Thermo Scientific) mass spectrometer as described previously [11] in a positive ionization mode. The instrument was equipped with the NSI ionization source with the capillary voltage adjusted to 2.2 kV and drying gas temperature adjusted to 280 °C. Precursor ions were scanned in a range from 400 m/z to 1200 m/z with a maximum integration time of 80 ms. Ions filtered by the charge state from z = 2+ to z = 6+ were isolated by a quadrupole within ±1.5 m/z window and taken for dissociation using the HCD mode (high-energy collision-induced dissociation) at 27% of normalized energy. The ubiquitin mass tags of ΔM = 114.0429 u (corresponding to GG) and ΔM = 383.2281 u (corresponding to LRGG) were detected using the MS3 synchronous fragment ions selection mode with an asymmetric mass tolerance.