Reprosil aq pur

Phosphopeptide-enriched samples were analysed on a Q Exactive high-performance Quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) [24 ] connected to an Easy-nLC 1000 nanoflow HPLC system (Thermo Fisher Scientific). Peptides were separated on a 50 cm column with an inner diameter of 75 μm filled with 1.8 μm C18 beads (Reprosil-AQ Pur, Dr. Maisch GmbH, Ammerbuch, Germany) prepared as described [25 ]. Peptides were eluted with acetonitrile in 0.1% formic acid using a gradient of 5-30% acetonitrile in 95min, 30-60% in 30 min and 60-95% in 8 min at a flow of 250 nl/min and a column temperature of 50°C [25 ]. Mass spectra were acquired in a data-dependent manner by automatically switching between MS and MS/MS in a top 10 approach. The resolution was 70000 for full spectra and 17500 (both at m/z 200) for HCD-derived fragments. The dynamic exclusion time was 30 sec.

LC-MS/MS was carried out by nanoflow reverse-phase liquid chromatography (Dionex Ultimate 3000, Thermo Scientific) coupled online to a Q-Exactive HF Orbitrap mass spectrometer (Thermo Scientific), as reported previously (Ni et al., 2019 (link)). Briefly, the LC separation was performed using a PicoFrit analytical column (75 μm ID×50 cm long, 15 µm Tip ID; New Objectives) in-house packed with 3-µm C18 resin (Reprosil-AQ Pur, Dr. Maisch).

The samples were separated on a 50 cm PicoFrit column (75 μm inner diameter) in-house packed with 1.9 μm C₁₈ beads (Reprosil-AQ Pur, Dr. Maisch) on an EASY-nLC 1000 system connected to a Q-Exactive HF (Thermo Scientific, Bremen, Germany). The peptides were separated with a gradient going from 2% to 25% buffer B in 110 min followed by a 25 min step to 40%. After the gradient the column was washed by going to 60% in 5 min, held for 5 min and re-equilibrated back to 2% for 15 min, resulting in a final acquisition of 165 min. Buffers contained 0.1% TFA dissolved in either 80% acetonitrile for buffer B, or milli-Q water for buffer A. The flow rate was 200 nL/min throughout the gradient and wash.
The Q-Exactive HF was operated in data-dependent top 10 mode. Full scan mass spectra were recorded at a resolution of 120,000 at m/z 200 over the m/z range 300–1750 with a target value of 3e6 and a maximum injection time of 20 ms. HCD-generated product ions were recorded with a maximum ion injection time set to 108 ms through a target value set to 2e5 and recorded at a resolution of 60,000 with a fixed first mass set to m/z 100. Normalized collision energy was 28%. The isolation window was set at 1.3 m/z units and the dynamic exclusion to 30 s.