Seppak cartridge

For TMT Labeling and high pH reversed‐phase chromatography, the samples were labeled with tandem mass tag (TMT) multiplex reagents according to the manufacturer’s protocol (Thermo Fisher Scientific, Loughborough, UK) and the labeled samples were pooled. The pooled sample was then desalted using a SepPak cartridge according to the manufacturer’s instructions (Waters, Milford, Massachusetts, USA). Eluate from the SepPak cartridge was evaporated to dryness and resuspended in buffer A (20 mM ammonium hydroxide, pH 10) prior to fractionation by high pH reversed‐phase chromatography using an Ultimate 3,000 liquid chromatography system (Thermo Fisher Scientific). In brief, the sample was loaded onto an XBridge BEH C18 Column (130 Å, 3.5 µm, 2.1 mm × 150 mm, Waters, UK) in buffer A and peptides eluted with an increasing gradient of buffer B (20 mM ammonium hydroxide in acetonitrile, pH 10) from 0% to 95% over 60 min. The resulting fractions were evaporated to dryness and resuspended in 1% formic acid prior to analysis by nano‐LC MSMS using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific).

An equal volume of each protein sample was digested overnight at 37 °C with 2.5 µg trypsin, labelled with TMT ten plex reagents according to the manufacturer’s protocol (Thermo Fisher Scientific), and the labelled samples pooled. An aliquot of the pooled sample was evaporated to dryness, resuspended in 5% formic acid and then desalted using a SepPak cartridge according to the manufacturer’s instructions (Waters, USA). Eluate from the SepPak cartridge was again evaporated to dryness and resuspended in buffer A (20 mM ammonium hydroxide, pH 10) prior to fractionation by high pH reversed-phase chromatography using an Ultimate 3000 liquid chromatography system (Thermo Scientific). In brief, the sample was loaded onto an XBridge BEH C18 Column (130 Å, 3.5 µm, 2.1 mm × 150 mm, Waters, UK) in buffer A and peptides eluted with an increasing gradient of buffer B (20 mM ammonium hydroxide in acetonitrile, pH 10) from 0–95% over 60 min. The resulting fractions were evaporated to dryness and resuspended in 1% formic acid prior to analysis by nano-LC MSMS using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific).

Pull-down samples were reduced (10 mM TCEP 55 °C, 1 h), alkylated (18.75 mM iodoacetamide, room temperature, 30 min), digested on the beads with trypsin (2.5 μg trypsin; 37 °C, overnight), and then labelled with Tandem Mass Tag (TMT) six-plex reagents according to the manufacturer's protocol (Thermo Fisher Scientific, Loughborough LE11 5RG, UK), and the labelled samples were pooled.
The pooled sample was evaporated to dryness, resuspended in 5% formic acid, and then desalted using a SepPak cartridge according to the manufacturer's instructions (Waters, Milford, Massachusetts, USA). The eluate from the SepPak cartridge was again evaporated to dryness and resuspended in buffer A (20 mM ammonium hydroxide, pH 10) prior to fractionation by high pH reversed-phase chromatography using an UltiMate 3000 liquid chromatography system (Thermo Fisher Scientific). In brief, the sample was loaded onto an XBridge BEH C18 Column (130 Å, 3.5 μm, 2.1 mm × 150 mm, Waters, UK) in buffer A and peptides eluted with an increasing gradient of buffer B (20 mM Ammonium Hydroxide in acetonitrile, pH 10) from 0% to 95% over 60 min. The resulting fractions (4 in total) were evaporated to dryness and resuspended in 1% formic acid prior to analysis by nano-LC MSMS using an Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Scientific).