Both unfractionated samples were each analyzed 20 times consecutively on an Orbitrap Fusion Lumos mass spectrometer operated in positive-mode with a Proxeon EASY-nLC 1200 liquid chromatograph (Thermo Fisher Scientific) as described previously.2 (link) Peptide fractionation was performed on a 100 μm inner diameter microcapillary column packed with 35 cm of Accucore C18 resin (2.6 μm, 150 Å, Thermo Fisher Scientific). Approximately 1 μg of peptide was loaded onto the column for LC–MS/MS analysis.
Separation occurred across a 90 min gradient from 4% to 35% acetonitrile in 0.125% formic acid. The flow rate was set to 525 nL/min over the gradient. To prevent carry over, the 20 analyses of the human only sample (H) were queued first followed by the 20 analyses of the human + 10% yeast spike-in sample (HY).
A data dependent Top Speed (3 s) method was used to collect spectra: high resolution MS1 spectra (Orbitrap resolution: 120 000; mass range: 350–1400 Th; and automatic gain control (AGC) target: 4 × 105; maximum injection time 50 ms) and high resolution MS2 spectra (Quadrupole isolation window: 1.6 Th; Orbitrap resolution: 7500; HCD energy: 30%; AGC target: 5 × 104; maximum injection time: 22 ms). Dynamic exclusion was enabled with a duration time of 120 s.
Separation occurred across a 90 min gradient from 4% to 35% acetonitrile in 0.125% formic acid. The flow rate was set to 525 nL/min over the gradient. To prevent carry over, the 20 analyses of the human only sample (H) were queued first followed by the 20 analyses of the human + 10% yeast spike-in sample (HY).
A data dependent Top Speed (3 s) method was used to collect spectra: high resolution MS1 spectra (Orbitrap resolution: 120 000; mass range: 350–1400 Th; and automatic gain control (AGC) target: 4 × 105; maximum injection time 50 ms) and high resolution MS2 spectra (Quadrupole isolation window: 1.6 Th; Orbitrap resolution: 7500; HCD energy: 30%; AGC target: 5 × 104; maximum injection time: 22 ms). Dynamic exclusion was enabled with a duration time of 120 s.
