A total of 110 MS runs
were analyzed as previously described27 (link) using an UltiMate 3000 Nano LC system coupled to an Orbitrap Fusion
Lumos Tribrid mass spectrometer (Thermo Fisher Scientific, San Jose).
SCX–SD–SEC fractions with large sample amounts were
injected as technical duplicates. Briefly, mobile phase A contained
0.1% (v/v) FA in water and mobile phase B contained 80% (v/v) ACN
and 0.1% (v/v) FA in water. Fractionated peptides were injected onto
a 500 mm C-18 EasySpray column (75 μm ID, 2 μm particles,
100 Å pore size) and separated using a constant flow rate of
250 nL/min. Depending on the sample amount per fraction, a linear
gradient from 4 to 40% mobile phase B was employed for either 60 or
139 min for peptide elution. MS1 spectra were acquired at 120 000
resolution in the orbitrap with an AGC target of 2 × 105 ions and a maximum injection time of 50 ms. For fragmentation, precursor
ions with charge states 3–8 and an intensity higher than 5
× 104 were isolated using an isolation window of 1.4 m/z (AGC target, 1–5 × 104; 60 ms max. injection time). Depending on the charge state
and the m/z ratio, precursor ions
were fragmented with energies based on the optimized data-dependent
decision tree using HCD/EThcD fragmentation.27 (link) MS2 spectra were recorded at 30 000 resolution in the orbitrap.
were analyzed as previously described27 (link) using an UltiMate 3000 Nano LC system coupled to an Orbitrap Fusion
Lumos Tribrid mass spectrometer (Thermo Fisher Scientific, San Jose).
SCX–SD–SEC fractions with large sample amounts were
injected as technical duplicates. Briefly, mobile phase A contained
0.1% (v/v) FA in water and mobile phase B contained 80% (v/v) ACN
and 0.1% (v/v) FA in water. Fractionated peptides were injected onto
a 500 mm C-18 EasySpray column (75 μm ID, 2 μm particles,
100 Å pore size) and separated using a constant flow rate of
250 nL/min. Depending on the sample amount per fraction, a linear
gradient from 4 to 40% mobile phase B was employed for either 60 or
139 min for peptide elution. MS1 spectra were acquired at 120 000
resolution in the orbitrap with an AGC target of 2 × 105 ions and a maximum injection time of 50 ms. For fragmentation, precursor
ions with charge states 3–8 and an intensity higher than 5
× 104 were isolated using an isolation window of 1.4 m/z (AGC target, 1–5 × 104; 60 ms max. injection time). Depending on the charge state
and the m/z ratio, precursor ions
were fragmented with energies based on the optimized data-dependent
decision tree using HCD/EThcD fragmentation.27 (link) MS2 spectra were recorded at 30 000 resolution in the orbitrap.
