Liquid chromatography-mass spectrometry (LC-MS) analysis
Tryptic peptides were separated using an Ultimate 3000 RSLCnano LC
system (Thermo Fisher Scientific; Waltham, US) equipped with a PEPMAP100
C18 5 µm 0.3 x 5 mm trap (Thermo Fisher Scientific; Waltham, US) and an
HSS-T3 C18 1.8 μm, 75 μm x 250 mm analytical reversed-phase column
(Waters Corporation; Milford, US). Mobile phase A was water containing
0.1% (v/v) formic acid and 3% (v/v) DMSO. Peptides were separated by
running a gradient of 2–35% mobile phase B (0.1% (v/v) formic acid,
3% (v/v) DMSO in ACN) over 40 min at a flow rate of 300 nl/min. Total
analysis time was 60 min including wash and column re-equilibration
steps. Column temperature was set to 55°C. Mass spectrometric analysis
of eluting peptides was conducted on an Orbitrap Exploris 480 (Thermo
Fisher Scientific; Waltham, US) instrument platform. Spray voltage was
set to 1.8 kV, the funnel RF level to 40, and heated capillary
temperature was at 275°C. Data were acquired in data-dependent
acquisition (DDA) mode targeting the 10 most abundant peptides for
fragmentation (Top10). Full MS resolution was set to 120,000 atm/z 200 and full MS automated gain control (AGC) target to 300%
with a maximum injection time of 50 ms. Mass range was set to m/z350–1,500. For MS2 scans, the collection of isolated peptide precursors
was limited by an ion target of 1 × 105 (AGC tar-get
value of 100%) and maximum injection times of 25 ms. Fragment ion
spectra were acquired at a resolution of 15,000 at m/z 200.
Intensity threshold was kept at 1E4. Isolation window width of the
quadrupole was set to 1.6 m/z and normalized collision energy was
fixed at 30%. All data were acquired in profile mode using positive
polarity. Each sample was analyzed in three technical replicates.