Es802 column

Samples were prepared by immunoprecipitated HEK293T cells expressing HA-NLGN4X, HA-NLGN4Y, and using untransfected HEK293T as control (n=3). Samples were subjected to trypsin digestion after TECP reduction and NEM alkylation. Digests were extracted from the gel and desalted using Waters Oasis HLB μElution plate. LC-MS/MS data acquisition was performed in data-dependent acquisition (DDA) mode on an Orbitrap Lumos mass spectrometer (Thermo Fisher Scientific) coupled with a 3000 Ultimate high pressure liquid chromatography instrument (Thermo Fisher Scientific). Peptides were separated over a 62-min gradient (5% – 35% MPB) on a ES802 column (Thermo Fisher Scientific) at a flow rate of 300 nl/min. The MS resolution is 120K at m/z 400, MS scan range is 300–1500 m/z, the automated gain control (AGC) target is 2 × 10e5. The quadrupole isolation window is 1.4 m/z. Precursors with charge states 2–6 and intensity higher than 1×10e4 within a 3 sec cycle between MS1 scans are selected for MS/MS acquisition in the linear ion trap, and activated with higher energy collisional dissociation (HCD) method.

The concentrated peptide mix was reconstituted in a solution of 2% acetonitrile (ACN) and 2% formic acid for MS analysis. Peptides were eluted from the ES802 column (75 μm × 25 cm; Thermo Fisher) at 300 nl/min using a Dionex UltiMate 3000 nano LC system with a 90-min gradient from 2 to 35% buffer B (100% acetonitrile and 0.1% formic acid). The gradient was switched from 35 to 85% buffer B over 1 min and held constant for 2 min. Last, the gradient was changed from 85% buffer B to 98% buffer A (100% water and 0.1% formic acid) over 1 min, and then held constant at 98% buffer A for five more minutes. The application of a 2.0-kV distal voltage electrosprayed the eluting peptides directly into the Thermo Fusion Tribrid mass spectrometer equipped with an EASY-Spray source (Thermo Scientific). Mass spectrometer-scanning functions and HPLC (high-performance LC) gradients were controlled by the Xcalibur data system (Thermo Fisher Scientific). MS data were acquired in the Orbitrap Fourier Transform (FT) at 120,000 resolutions from m/z (mass/charge ratio) 400 to 1600. Collision-induced dissociation MS/MS was acquired in the ion trap on 2⁺ and higher charge state ions for 3-s duty cycles.