Picoview nanospray interface

For mass spectrometry analysis, LC was performed on an Agilent 1100 series HPLC system (Agilent Technologies, USA) with a micro-T for flow splitting coupled to an LTQ-Orbitrap XL hybrid mass spectrometer (Thermo Electron, USA) equipped with a PicoView nanospray interface (New Objective, USA). Peptide mixtures were loaded onto a 75 μm × 250 mm fused silica capillary column packed in-house with C18 resin (5 μm, Nucleosil 120–5 C18; Macherey-Nagel, Germany), and were separated over 110 min using a linear gradient of 2–50 % solvent B (95 % acetonitrile with 0.1 % formic acid) and solvent A (0.1 % formic acid in water) at a flow rate of 300 nL/min. The LTQ-Orbitrap was operated in positive ion mode and mass spectra from full scans were acquired on the Orbitrap analyzer (m/z 350–1600) with resolution set to R = 30,000 (at m/z 400). The ten most intense peptide ions were selected from the MS scan and fragmented by collision-induced dissociation for MS/MS scan in the LTQ analyzer. All Orbitrap measurements were performed with the “lock mass” option to improve mass accuracy of precursor ions.

NanoLC-nanoESI-MS/MS analysis was performed on a nanoAcquity system (Waters, MA, USA) connected to the Orbitrap Elite hybrid mass spectrometer (Thermo Electron, Bremen, Germany) equipped with a PicoView nanospray interface (New Objective, MA, USA). Peptide mixtures were loaded onto a 75 μm inner diameter, 25 cm length C18 BEH column (Waters) packed with 1.7 μm particles with a pore width of 130 Å and separated using a segmented gradient in 180 min from 5% to 40% solvent B (acetonitrile with 0.1% formic acid) at a flow rate of 300 nl/min and a column temperature of 35 °C. Solvent A was 0.1% formic acid in water. The mass spectrometer was operated in the data-dependent mode. Briefly, survey full scan MS spectra were acquired in the Orbitrap (m/z 350–1600) with the resolution set to 120K at m/z 400 and automatic gain control target at 10⁶. The 15 most intense ions were sequentially isolated for higher-energy collisional dissociation (HCD) MS/MS fragmentation and detection in the Orbitrap with previously selected ions dynamically excluded for 60 s. For MS/MS, we used a resolution of 15,000, an isolation window of 2 m/z, and a target value of 50,000 ions, with maximum accumulation times of 200 ms. Fragmentation was performed with normalized collision energy of 35% and an activation time of 0.1 ms. Ions with singly and unrecognized charge state were also excluded.

In order to compile a comprehensive phosphoproteome data set, the total protein was treated with trypsin in both gel-based and gel-free processes following procedures described in the literature57 (link). Phosphopeptides from the tryptic peptides were enriched by custom-made HAMMOC tips, which were prepared using 0.5 mg TiO₂ beads (GL Sciences, Tokyo, Japan) packed into 10-μL C₈-StageTips, as described previously29 (link)30 (link).
The peptide mixtures were analyzed by online nanoflow liquid chromatography tandem mass spectrometry (LC-MS/MS) on a nanoAcquity system (Waters, Milford, MA) coupled to an LTQ-Orbitrap Velos hybrid mass spectrometer (Thermo Scientific) equipped with a PicoView nanospray interface (New Objective). Detailed detection conditions are described in Supplementary Information.