Easy spray ion source

Peptide samples were analyzed by LC-MS/MS on a Q Exactive mass spectrometer (Thermo Fisher Scientific) coupled to an EASY-nLC 1000 liquid chromatography system (Thermo Scientific) via an EASY-Spray ion source (Thermo Fisher Scientific). Peptides were fractionated on a 75 μm × 500 mm EASY-Spray column (Thermo Scientific) over various gradient lengths from 90 min to 240 min. The following describes the typical analytical set-up, but further specific details of MS run conditions can be found within the raw data files. Precursor ion full scan spectra were acquired over (m/z 300 to 1,800) with a resolution of 70,000 at m/z 200 (target value of 1,000,000 ions, maximum injection time 20 ms). Up to ten data dependent MS² spectra were acquired with a resolution of 17,500 at m/z 200 (target value of 500,000 ions, maximum injection time 60 ms). Ions with unassigned charge state, and singly or highly (>8) charged ions were rejected. Intensity threshold was set to 2.1 × 10⁴ units. Peptide match was set to preferred, and dynamic exclusion option was enabled (exclusion duration 40 s). The mass spectrometry proteomics raw data files have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (http://www.ebi.ac.uk/pride/archive/) with the dataset identifier PXD003530 for the label-free site ID analysis, and PXD004995 for the occupancy and SILAC half-life analysis.

Tandem mass tag (TMT)‐labeled peptides (1 μg) from each of the 24 fractions were dissolved in solvent A (2% acetonitrile and 0.1% formic acid); solvent B consisted of 98% acetonitrile and 0.1% formic acid. Nano‐LC‐MS/MS analyses were performed using a Q Exactive Mass Spectrometer (Thermo Scientific) equipped with an EASY‐Spray Ion Source and coupled to an EASY‐nLC 1000 (Thermo Scientific). Detailed methods are provided in supplementary material and methods sections.

A Dionex UltiMate 1000 system (Thermo Fisher Scientific) was coupled to an Orbitrap Fusion Lumos (Thermo Fisher Scientific) through an Easy-Spray ion source (Thermo Fisher Scientific). Peptide samples were dissolved in 2% acetonitrile/0.1% formic acid (20 µl), loaded (19 µl, 15 µl/min, 3 min) onto a trap column (100 µm × 2 cm, 5 µm Acclaim PepMap 100 C18, 50°C), eluted (0.200 µl/min) onto an Easy-Spray PepMap RSLC C18 column (2 µm, 50 cm × 75 µm inner diameter, 50°C; Thermo Fisher Scientific), and separated with the following gradient, all percentages of buffer B (0.1% formic acid in acetonitrile): 0–110 min, 2–22%; 110–120 min, 22–35%; 120–130 min, 35–95%; 130–150 min, isocratic at 95%; 151–153 min, 95–2%; 153–171 min, isocratic at 2%. Spray voltage was 1,900 V, ion transfer tube temperature was 275°C, and radio frequency lens was 30%. MS scans were acquired in profile mode and MS/MS scans in centroid mode, for ions with charge states 2–5, with a cycle time of 3 s. MS spectra were recorded from 375 to 1,500 daltons at 120,000 resolution (at mass to charge 200), and higher energy collision-induced dissociation MS/MS was triggered above a threshold of 2.0e4, with quadrupole isolation (0.7 daltons) at 30,000 resolution, and collision energy of 30%. Dynamic exclusion was used (±5 parts per million, 60 s), and monoisotopic precursor selection was on.

The MS analysis was performed on a 50 cm EASY-Spray column connected online to a Q Exactive HF instrument through an EASY-Spray™ Ion Source (Thermo Fisher Scientific, Waltham, MA, USA). Solvent A was 0.1% formic acid (FA) in ddH₂O and solvent B was 80% ACN plus 0.1% FA. Peptides were injected in an aqueous 1% trifluoroacetic acid (TFA) solution at a flow rate of 500 nL/min and were separated with a 95 min 3%–60% gradient of solvent B (80 min 3–30%, 10 min 30–40%, 5 min 40–60%), at a flow rate of 250 nL/min. The Q Exactive HF instrument was operated in the data-dependent acquisition (DDA) mode to automatically switch between full scan MS and MS/MS acquisition. Survey full scan MS spectra (m/z 375–1650) were analyzed in the Orbitrap detector with a resolution of 60,000 at m/z 200. The 10 most intense peptide ions with charge states comprised between 2 and 4 were sequentially isolated to a target value for MS1 of 3 × 10⁶ and fragmented by higher-energy collisional dissociation (HCD) with a normalized collision energy setting of 28%. The maximum allowed ion accumulation times were 20 ms for full scans and 80 ms for MS/MS, and the target value for MS/MS was set to 1 × 10⁵. The dynamic exclusion time was set to 20 s, and the standard mass spectrometric conditions for all experiments were as follows: spray voltage of 1.8 kV, no sheath and auxiliary gas flow.