U3000 rslcnano

The dried peptide samples were reconstituted in 5% acetonitrile (ACN) with 0.1% formic acid (FA) and subsequently loaded using a nano-HPLC (Dionex U3000 RSLCnano) onto a PepMap100 loading column (C18, L = 20 mm, 3 µm particle size, Dionex) and washed with loading buffer (2% ACN, 0.05% trifluoroacetic acid (TFA) in water) for 6 min at a flow rate of 6 µL/min. Peptides were separated on a PepMap RSLC analytical column (C18, L = 50 cm, <2 µm particle size, Dionex) by a gradient of phase A (water with 5% v/v dimethylsulfoxide [DMSO] and 0.1% FA) and phase B (5% DMSO, 15% water and 80% ACN v/v/v). The gradient was ramped from 4% B to 48% B in 178 min at a flow rate of 300 nL/min. All solvents were LC-MS grade and purchased from Fluka. Eluting peptides were ionized online using a Nanospray Flex ion source (Thermo Scientific) and analyzed either in a QExactive Plus (Thermo Scientific) or in a Lumos Fusion (Thermo Scientific) mass spectrometer in data-dependent acquisition mode. The full parameter sets are listed in Supplementary file 2. All fractions of the ‘triple SILAC’ samples were measured in technical duplicates. For ‘double SILAC’ samples, one technical replicate was measured.

The dried peptide fractions were dissolved in 5% acetonitrile with 0.1% formic acid, and subsequently loaded using a nano-HPLC (Dionex U3000 RSLCnano) onto a PepMap100 trapping column (C₁₈, particle size 3 µm, L = 20 mm). Peptides were separated on a PepMap RSLC analytical column (C₁₈, particle size <2 µM, L = 50 cm, Dionex/Thermo Fisher Scientific) by a gradient of water (buffer A: water with 5% v/v dimethylsulfoxide and 0.1% formic acid) and acetonitrile (buffer B: 5% dimethylsulfoxide, 15% water and 80% acetonitrile (v/v/v), and 0.08% formic acid), running from 4% to 48% B in 178 min at a flowrate of 300 nL/min. All LC-MS-grade solvents were purchased from Fluka.
Peptides eluting from the column were ionized online using a Thermo nanoFlex ESI source and analyzed in a ‘Q Exactive Plus’ mass spectrometer (Thermo Fisher Scientific). Mass spectra were acquired over the mass range 350–1,400 m/z, and sequence information were acquired by data-dependent automated switching to MS/MS mode using collision energies based on mass and charge state of the candidate ions (TOP12, MS resolution 70 k, MS/MS resolution 35 k, injection time: 120 ms, full parameters in Supplementary file 8). All samples were measured in quadruplicate LC-MS/MS runs.

Liquid chromatography-tandem
mass spectrometry (LC-MS/MS) proteomic analysis was performed following
the methods previously reported^{49 (link)} with
modification. Dried peptide pellets were dissolved in 50 μL
of loading buffer, consisting of 3% acetonitrile and 0.1% trifluoroacetic
acid in water, and sonicated in a water bath for 3 min for full suspension,
after which they were cleared by centrifugation at 13,000 rpm for
2 min. LC-MS/MS was performed and analyzed by nanoflow liquid chromatography
(U3000 RSLCnano, Thermo Fisher Scientific, United Kingdom) coupled
to a hybrid quadrupole-orbitrap mass spectrometer (Q Exactive HF,
Thermo Fisher Scientific, United Kingdom). Peptides were separated
on an Easy-Spray C18 column (75 μm × 50 cm) using a 2-step
gradient from 3% solvent A (0.1% formic acid in water) to 10% B over
5 min and then to 50% solvent B (0.1% formic acid in 80% acetonitrile)
over 75 min at 300 nL min^–1, 40 °C. The mass
spectrometer was programmed for data-dependent acquisition with 10
product ion scans (resolution 30,000, automatic gain control 1 ×
10⁵, maximum injection time 60 ms, isolation window 1.2
Th, normalized collision energy 27, and intensity threshold 3.3 ×
10⁴) per full MS scan (resolution 120,000, automatic gain
control 1 × 10⁶, maximum injection time 60 ms) with
a 20 s exclusion time.