C18 resin

The peptide mixtures were desalted using C18 resin (The Nest Group) according to the manufacturer’s protocol. LC-MS/MS analyses were performed on a Q-Exactive Plus high-resolution mass spectrometer (Thermo Scientific, Inc.) with a nanoAcquity UPLC system (Waters Corp.) and a nano-electrospray ionization source. Samples were trapped on a Symmetry C18 300 mm × 180 μm trapping column for 3 min at 5 μL/min (99.9/0.1 v/v water/acetonitrile 0.1% formic acid), and separated on a 75 μm × 250 mm column packed with 1.7 μm Acquity HSST3 C18 stationary phase (Waters Corp.). Peptides were separated using a gradient of 3 to 30% acetonitrile with 0.1% formic acid over 90 min at a flow rate of 0.4 μL/min. Data collection was performed in a data-dependent acquisition (DDA) mode with a resolution of 70,000 (at m/z 200) for full MS scan from m/z 375 ~ 1600 with a target AGC value of 1 × 10⁶ ions, followed by 20 product ion scans at a resolution of 17,500 (at m/z 200), using an AGC target value of 1 × 10⁵ ions, a max fill time of 60 ms, and normalized collision energy of 30 V. Each sample was analyzed in triplicate.

The protein samples generated in the SILAC-LiP and SILAC-SPROX experiments were treated with tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (ThermoFisher) (5 mM) for 1 h at 60 °C. The proteins were reacted with 10 mM methylmethane thiosulfonate (MMTS) (Sigma-Aldrich) at room temperature for 15 min to block cysteine side chains. The SILAC-LiP samples were diluted with 0.5 M triethylammonium bicarbonate (TEAB) buffer (Sigma) such that the final concentration of GdmCl was less than 2 M. Trypsin was added to each of the SILAC-LiP and SILAC-SPROX samples. In all cases, the ratio of trypsin to total protein was 1:20 to 1:50. The trypsin digestions were allowed to proceed at 37 °C for 16–18 h before quenching with 10 % trifluoracetic acid (TFA) to pH 2–3. The digested samples were desalted using C18 resin (The Nest Group) according to the manufacturer’s protocol.

The tryptic peptides were desalted using C18 resin (The Nest Group, Southborough, MA) according to the manufacturer’s protocol and analyzed by liquid-chromatography-tandem MS (LC-MS/MS) using an EASY-nLC 1000 system coupled to a Q-Exactive (quadrupole-orbitrap) mass spectrometer (Thermo Fisher Scientific). Columns were packed in-house with Jupiter 4µ Proteo 90Å reversed phase resin (Phenomenex). Peptides were concentrated and desalted on a trapping column (100µm ID x 20 mm) and eluted on an analytical column (75µm ID x 150 mm), operating at 300 nl/min and using the following gradient: 5% B for 3 min, 5–35% B in 120 min, 35–80% B in 2 min, and 80% B for 9 min [solvent A: 0.1% formic acid (v/v); solvent B: 0.1% formic acid (v/v), 80% CH₃CN (v/v) (Fisher Scientific)]. The Q-Exactive was operated in a data-dependent MS/MS mode using the top 10 most intense precursors detected in a survey scan from 300 to 1,800 m/z performed at 70K resolution. Tandem MS was performed by HCD fragmentation with stepped normalized collision energy (NCE) of 20%.