Nanoflex ion source

Dried peptides were reconstituted in 2% acetonitrile containing 0.1% TFA and analysed on a QExactive HF mass spectrometer coupled to an easy nLC 1200 (Thermo Fisher Scientific) fitted with a 35-cm, 75-µm ID fused-silica column packed in house with 1.9-µm C18 particles (Reprosil pur, Dr. Maisch). The column was maintained at 50 °C using an integrated column oven (Sonation). Peptides were eluted in a non-linear gradient of 4–28% acetonitrile over 45 min and directly sprayed into the mass spectrometer equipped with a nanoFlex ion source (Thermo Fisher Scientific). Full-scan MS spectra (300–1,650 m/z) were acquired in profile mode at a resolution of 60,000 at m/z 200, a maximum injection time of 20 ms and an AGC (automatic gain control) target value of 3 × 10⁶. Up to 15 of the most intense peptides per full scan were isolated using a 1.4-Th window for fragmentation by higher energy collisional dissociation (normalized collision energy of 28). MS/MS spectra were acquired in centroid mode with a resolution of 30,000, a maximum injection time of 45 ms and an AGC target value of 1 × 10⁵. Single charged ions, ions with a charge state of more than four and ions with unassigned charge states were not considered for fragmentation, and dynamic exclusion was set to 20 s to minimize the acquisition of fragment spectra representing already acquired precursors.

Samples were analyzed on a Q Exactive HF coupled to an easy nLC 1200 (ThermoFisher Scientific) using a 35 cm long, 75 µm ID home-made fused-silica emitter packed with 1.9 µm C18 particles (Reprosil pur, Dr. Maisch), and kept at 50°C using an integrated column oven (Sonation). Peptides were eluted by a linear gradient from 4-32% acetonitrile over 60 min and directly sprayed into the mass-spectrometer equipped with a nanoFlex ion source (Thermo Fisher Scientific). Full scan MS spectra (350–1650 m/z) were acquired in Profile mode at a resolution of 60,000 at m/z 200, a maximum injection time of 20 ms and an AGC target value of 3×10⁶ charges. Up to 10 peptides per full scan were isolated using a 1.4 Th window and fragmented using higher energy collisional dissociation (normalized collision energy of 27). MS/MS spectra were acquired in centroid mode with a resolution of 30,000, a maximum injection time of 54 ms and an AGC target value of 10⁵. Singly charged ions, ions with a charge state above 5 and ions with unassigned charge states were not considered for fragmentation and dynamic exclusion was set to 20 s to minimize selection of already fragmented precursors.

Peptides were resuspended in 0.1% FA and separated on an Easy nLC 1200 (ThermoFisher Scientific, USA) and a 22-cm-long, 75-mm ID fused-silica column, which had been packed in-house with 1.9 mm C18 particles (ReproSil-Pur, Dr. Maisch, Germany), and kept at 45°C using an integrated column oven (Sonation, Biberach, Germany). Peptides were eluted by a nonlinear gradient from 5% to 38% acetonitrile over 120 min and directly sprayed into a QExactive HF mass spectrometer equipped with a nanoFlex ion source (ThermoFisher Scientific, USA) at a spray voltage of 2.3 kV. Full-scan MS spectra (350–1,400 m/z) were acquired at a resolution of 120,000 at m/z 200, a maximum injection time of 100 ms, and an AGC target value of 33,106. Up to 20 most intense peptides per full scan were isolated using a 1-Th window and fragmented using higher-energy collisional dissociation (normalized collision energy of 35). MS/MS spectra were acquired with a resolution of 45,000 at m/z 200, a maximum injection time of 80 ms, and an AGC target value of 13,105. Ions with charge states of 1 and > 6, as well as ions with unassigned charge states, were not considered for fragmentation. Dynamic exclusion was set to 20 s to minimize repeated sequencing of already-acquired precursors.

Unless stated otherwise, peptides were resuspended in 0.1% FA and separated on an easy nLC 1200 (ThermoFisher Scientific) and a 22 cm long, 75 μm ID fused-silica column, which has been packed in house with 1.9 μm C18 particles (ReproSil-Pur, Dr. Maisch), and kept at 45 °C using an integrated column oven (Sonation). Peptides were eluted by a non-linear gradient from 5 to 38% acetonitrile over 120 min and directly sprayed into a QExactive HF mass-spectrometer equipped with a nanoFlex ion source (ThermoFisher Scientific) at a spray voltage of 2.3 kV. Full-scan MS spectra (350–1400 m/z) were acquired at a resolution of 120,000 at m/z 200, a maximum injection time of 100 ms and an AGC target value of 3 × 10⁶. Up to 20 most intense peptides per full scan were isolated using a 1 Th window and fragmented using higher energy collisional dissociation (normalized collision energy of 35). MS/MS spectra were acquired with a resolution of 45,000 at m/z 200, a maximum injection time of 80 ms and an AGC target value of 1 × 10⁵. Ions with charge states of 1 and > 6 as well as ions with unassigned charge states were not considered for fragmentation. Dynamic exclusion was set to 20 s to minimize repeated sequencing of already acquired precursors.

Dried peptides were resuspended with 0.5 µg/µl in 2 % (v/v) acetonitrile / 1 % (v/v) formic acid solution. Samples were shot with settings similar to previously studies^{65 (link)}. First, peptides were separated on an Easy nLC 1200 (ThermoFisher Scientific) and a 22 cm long, 75 mmID fused-silica column, which had been packed in house with 1.9 mm C18 particles (ReproSil-Pur, Dr. Maisch), and kept at 45-50 °C using an integrated column oven (Sonation). Peptides were eluted by a non-linear gradient from 5%–38% acetonitrile over 120 min and subsequently sprayed into a QExactive HF mass spectrometer equipped with a nanoFlex ion source (ThermoFisher Scientific) at a spray voltage of 2.3 kV. Full scan MS spectra (350–1400 m/z) were acquired at a resolution of 120,000 at m/z 200, a maximum injection time of 100 ms and an AGC target value of 3 × 10⁶. Up to 20 most intense peptides per full scan were isolated using a 1 Th window and fragmented using higher energy collisional dissociation (normalized collision energy of 35). MS/MS spectra were acquired with a resolution of 45,000 at m/z 200, a maximum injection time of 86 ms and an AGC target value of 1 × 10⁵. Ions with charge states of 1 and >6 as well as ions with unassigned charge states were not considered for fragmentation. Dynamic exclusion was set to 20 s to minimize repeated sequencing of already acquired precursors.