Nanolc ultra 2d system

Online Nano-RPLC was used on an Eksigent nanoLC-Ultra™ 2D System (AB SCIEX). The samples were re-suspended with Nano-RPLC buffer A (0.1% formic acid, 2% acetonitrile), loaded on a C18 nanoLC trap column (100 μm × 3 cm, C18, 3-μm particle size, 150 Å), and washed with Nano-RPLC buffer A at a flow rate of 2 μl/min for 10 min. A linear LC gradient profile was used to elute peptides from the analytical ChromXP C18 column (75 μm × 15 cm, C18, 3-μm particle size, 120 Å, Eksigent). The gradient began with 5% Nano-RPLC buffer B (0.1% formic acid, 98% acetonitrile) and rose to 35% within 70 min. Data acquisition was performed with a Triple TOF 5600 System (AB SCIEX, USA) fitted with a Nanospray III source (AB SCIEX, USA) and a pulled quartz tip as the emitter (New Objectives, USA). Data were acquired using an ion spray voltage of 2.5 kV, curtain gas of 30 PSI, nebulizer gas of 5 PSI, and an interface heater temperature of 150°C. For information dependent acquisition, survey scans were acquired in 250 ms, and as many as 35 product ion scans were collected if they exceeded a threshold of 150 counts per second (counts/s) with a 2+ to 5+ charge-state. The total cycle time was fixed to 2.5 s. A rolling collision energy setting was applied to all precursor ions for collision-induced dissociation.

S2 cells expressing PIG-B-Flag were washed with phosphate-buffered saline (PBS) and suspended in 1% formaldehyde for crosslinking. After 10 min, cells were washed three times with PBS containing 1.25 M glycine, sonicated in RIPA buffer and centrifuged. Supernatants were incubated with anti-DYKDDDDK tag antibody-conjugated magnetic beads (Wako Junyaku). After three washes with RIPA buffer, bound proteins were eluted with 0.1 M triethylamine (pH 11.5). The solutions were dried, resolved using 1% sodium deoxycholate (SDC), 2 M urea and 50 mM NH₄HCO₃, and incubated with trypsin at 37°C overnight. The solutions containing digested peptides were processed using phase-transfer methods to remove SDC. The obtained peptides were resolved with 0.1% formic acid and desalted using an Empore C18 disk (GL Science). The peptides were separated on a C18 reversed-phase column (75 µm×150 mm; ChromXP C18-CL 3 µm 120A; Eksigent) equipped with a nanoLC-Ultra 2D system (AB SCIEX). The masses of the eluted peptides were determined using a TripleTOF^® 5600 (AB SCIEX) mass spectrometer.

Protein extraction and digestion were performed according to the methods described before^{41 (link)}. The protein digests were separated using a 10 min elution gradient at a flow rate of 2 µL per min in an Eksigent nanoLC-Ultra 2D system (AB SCIEX). A C18 reversed phase chromatographic column (75 μm × 15 cm, 3 μm, 120 Å, ChromXP Eksigent) was used as the analytical column. MS/MS scan was performed by tripleTOF5600 system (AB SCIEX).