Pepmap 100 c18 trap column

The resulting phosphopeptide mixture was analyzed using an Easy-nLC 1000 coupled to a Fusion mass spectrometer (Thermo Fisher Scientific). The sample was loaded onto a PepMap 100 C18 trap column (75 μm id × 2 cm length; 3 μm, 100 Å, C18 resin; Thermo Fisher Scientific) at 2 μl/min for 15 min with 0.10% formic acid (volume fraction) followed by separation on an Acclaim PepMap RSLC 2 μm C18 EasySpray column (75 μm id × 25 cm length; Thermo Fisher Scientific) with temperature set at 45 °C. After loading, peptides were separated along a 90 min multistep analytical run with mobile phase A being 0.1% formic acid (volume fraction) and mobile phase B being acetonitrile with 0.1% formic acid (volume fraction). The gradient started with 4% B and progressed to 9% B at 28 min, 15% B at 56 min, 24% B at 74 min, 35% B at 79 min, and 95% B at 80 min through the end of the run. The flow rate was maintained at 200 nl/min for the entire analysis and data were acquired for the entire run.

The resulting phosphopeptide mixture was analyzed using an UltiMate 3000 Nano LC coupled to a Fusion Lumos mass spectrometer (Thermo Fisher Scientific). The sample was loaded onto a PepMap 100 C18 trap column (75 μm id × 2 cm length; 3 μm, 100 Å, C18 resin; Thermo Fisher Scientific) at 3 μl/min for 10 min with 2% acetonitrile (volume fraction) and 0.05% TFA (volume fraction) followed by separation on an Acclaim PepMap RSLC 2 μm C18 column (75 μm id × 25 cm length; Thermo Fisher Scientific) at 40 °C. After loading, peptides were separated along a 120 min two-step gradient of 5% to 27.5% mobile phase B (80% acetonitrile and 0.08% formic acid) over 105 min followed by a ramp to 40% mobile phase B over 15 min. Lastly, the gradient was ramped to 95% mobile phase B over 10 min, and held at 95% mobile phase B for 10 min before returning to 5% mobile phase B, all at a flow rate of 300 nl/min. Mobile phase A is 0.1% formic acid (volume fraction) in water. Data were acquired on the mass spectrometer from 10 min to 150 min.

The peptides were dissolved in 40 µL of a solution containing 0.05% TFA and 4% acetonitrile. Thereafter, 1 µL of each sample was applied to an Ultimate 3000 reversed-phase capillary nano liquid chromatography system connected to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). All samples were injected and concentrated on a PepMap100 C18 trap column (3 µm, 100 Å, 75 µm inner diameter (i.d.) × 20 mm, nanoViper; Thermo Scientific) equilibrated with 0.05% TFA in water. After switching the trap column inline, LC separations were performed on an Acclaim PepMap100 C18 capillary column (2 µm, 100 Å, 75 µm i.d. × 500 mm, nanoViper; Thermo Scientific, Waltham, MA, USA) at an eluent flow rate of 300 nL/min. Mobile phase A consisted of 0.1% (v/v) formic acid in water, whereas mobile phase B contained 0.1% (v/v) formic acid and 80% (v/v) acetonitrile in water. The column was pre-equilibrated with 5% mobile phase B, followed by an increase to 44% mobile phase B over 70 min. Mass spectra were acquired in a data-dependent mode, utilizing a single MS survey scan (m/z 300–1650) with a resolution of 60,000 and MS/MS scans of the 15 most intense precursor ions with a resolution of 15,000. The dynamic exclusion time was set to 20 s and the automatic gain control was set to 3 × 10⁶ and 1 × 10⁵ for the MS and MS/MS scans, respectively.

Data-dependent acquisitions (DDAs, unbiased peptide sampling)–the peptides were and analyzed using the Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) fronted with an EasySpray ion source, and coupled to an Easy-nLC1000 HPLC pump (Thermo Fisher Scientific). The peptides were separated using a dual column set-up: an Acclaim PepMap 100 C18 trap column, 75 μm × 20 mm; and a PepMap RSLC C18 EASY-Spray LC heated (45°C) column, 75 μm × 250 mm (Thermo Fisher Scientific). The gradient flow rate was 300 nl/min from 5 to 21% solvent B (acetonitrile/0.1% formic acid) for 75 min, 21 to 30 % Solvent B for 15 min, followed by 10 min of a “jigsaw wash,” alternating between 5 and 95 % Solvent B. Solvent A was 0.1% formic acid. The instrument was set to 120 K resolution, and the top N precursor ions in a 3 s cycle time (within a scan range of 375–1,500 m/z; isolation window, 1.6 m/z; ion trap scan rate, normal) were subjected to collision induced dissociation (collision energy 30%) for peptide sequencing (or MS/MS). Dynamic exclusion was enabled (60 s).

Reversed phase chromatographic separation of cross-linked peptide samples was performed using an EASY-nLC 1200 system coupled with a Q-Exactive High Field mass spectrometer (Thermo Scientific). The nano-flow LC system was configured with a 20 mm, 75 micron ID PepMap100 C18 trap column (Thermo Scientific), and a 15 cm, 50 micron ID PepMap RSLC C18 analytical column (Thermo Scientific) with an steel emitter. Mobile phase A consisted of 2% acetonitrile/0.1% formic acid and mobile phase B consisted of 80% acetonitrile/ 0.1% formic acid. Samples containing cross-linked peptide pairs were injected through the autosampler onto the trap column. Peptides were then separated using the following linear gradient steps at a flow rate of 300 nL/min: 2% B for 1 min, 2% B to 35% B over 100 min, 35% B to 40% B over 5 min, 40% B to 80% B over 1 min, held at 80% B for 4 min, 80% B to 2% B over 1 min and held at 2% B for 8 min. Separation was accomplished with a 40 min linear gradient from 2% to 40% solvent B at 300 nL/min. The mass spectrometer was operated with a PRM method with the following settings: a resolving power of 15,000 @ 200 m/z, ACG target of 2E5 ions, maximum ion time of 10 ms, isolation window of 1 m/z and a normalized collision energy of 27.

For LC-MS/MS analysis, we used a reversed-phase liquid chromatography (RP-LC) system (Ultimate 3000 nano LC) interfaced with an LTQ-Orbitrap hybrid mass spectrometer equipped with a nanoelectrospray ion source (all from Thermo Fisher Scientific). The RP-LC system comprised a C18 PepMap 100 trap column (length × inner diameter: 0.5 × 0.3 mm; Thermo Fisher Scientific) and a C18 tip column (length × inner diameter: 10 cm × 75 μm; particle diameter: 3 μm; Nikkyo Technos, Tokyo, Japan). Samples were loaded onto the trap column, washed with H₂O containing 0.1% formic acid (solvent A) to concentrate and desalt them, and eluted using 95% acetonitrile, 5% H₂O and 0.1% formic acid (solvent B). The 120-min LC gradient changed from 97.5% A/2.5% B to 77.5% A/22.5% B at 109 min, 65% A/35% B at 5 min, and finally 2% A/98% B at 2 min (at 0.3 μL/min). Eluted peptides were ionized by the electrospray and analyzed using the mass spectrometer (electrospray voltage: 1.8 kV; no sheath and auxiliary gas flow; capillary temperature: 250°C; collision energy: 35%; ion-selection threshold: 500 counts for MS/MS; Top N: 15; dynamic exclusion: 60 s).

The peptides obtained by in-solution and in-gel digestion were vacuum-dried and reconstituted in 100 μl of 2% FA. The digested peptides were separated by online reversed-phase chromatography on an Agilent 1290 Ultra-high-performance LC (UHPLC) or Dionex UltiMate 3000 (Thermo Fisher Scientific) coupled to a Thermo Fisher Scientific Orbitrap Fusion mass spectrometer. Peptides were separated using a Poroshell 120 EC-C18 2.7-μm analytical column (ZORBAX Chromatographic Packing, Agilent) and a C18 PepMap 100 trap column (5 mm × 300, 5 μm; Thermo Fisher Scientific). Samples were eluted over a 90-min gradient from 0% to 35% acetonitrile at a flow rate of 0.3 μl/min. Peptides were analyzed with a resolution setting of 60,000 in MS1. MS1 scans were obtained with a standard automatic gain control target, a maximum injection time of 50 ms, and a scan range of 350–2,000. The precursors were selected with a 3 m/z window and fragmented by sHCD and EThcD. The sHCD fragmentation included steps of 25%, 35%, and 50% normalized collision energies (NCE). EThcD fragmentation was performed with calibrated charge-dependent electron-transfer dissociation parameters and 27% NCE supplemental activation. For both fragmentation types, MS2 scans were acquired at a 30,000 resolution, a 4 × 10⁻⁵ automatic gain control target, a 250-ms maximum injection time, and a scan range of 120–3,500.