C18 macrospin column

Cells expressing the GFP-tagged SRMS variants were verified under a fluorescent microscope to ensure that equivalent and over 80–90% transfection efficiencies were achieved. The transfected cells were trypsinized, washed with 1X PBS and counted. 3 × 10⁶ cells, from each condition, were lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails (Pierce™, USA). For complete lysis, the cells were sonicated using three bursts of 10% amplitude followed by two bursts of 15% amplitude for 10 s each. The lysates were centrifuged at 12,000 r.c.f for 10 mins and the clarified lysates collected. Total proteins were purified by chloroform:methanol:water precipitation and the precipitated proteins resuspended in 8 M urea pH 8.0 containing 400 mM ABC. The proteins were reduced with 10 mM DTT and alkylated with 40 mM iodoacetamide followed by digestion at 37 °C with Lys-C (1:100 enzyme to protein ratio) for 6 h and trypsin (1:100 enzyme to protein ratio) overnight. The digestion reactions were quenched with 0.1% formic acid and desalted using C₁₈ MacroSpin columns (The Nest Group, USA). The desalted samples were dried in a speedvac and dissolved in a buffer comprising 3.5% formic acid and 0.1% trifluoroacetic acid (TFA).

After incubation at 98 °C samples were reduced for 1 hour at 37 °C with 5 mM tris(2-carboxyethyl)phosphine hydrochloride followed by a 30 min incubation at RT in the dark with 20 mM iodoacetamide. Subsequently, samples were digested for 2 hours at 37 °C with lysyl endopeptidase (1:100 enzyme: substrate ratio) in 2 additional volumes of 0.1 M ammonium bicarbonate (final pH of 8). Following this, samples were further digested for 16 hours at 37 °C with trypsin (1:100 enzyme: substrate ratio). Formic acid was added to a final concentration of 1.5% to precipitate the deoxycholate, the samples were centrifuged at 16,000 × g for 10 min and the supernatant was transferred to a new Eppendorf tube. An equal volume of formic acid was added again and the centrifugation step was repeated. Digests were desalted using C18 MacroSpin columns (The Nest Group) following the manufacturer’s instructions and after drying resuspended in 1% acetonitrile (ACN) and 0.1% formic acid. Biognosys’ iRT kit (Biognosys AG, Schlieren, Switzerland) was added to all samples according to the manufacturer’s instructions.

A 15‐cm dish of confluent cells was washed three times with PBS and then lysed by resuspension in 8 M urea and 0.1 M ammonium bicarbonate (to 1 μg/μL protein). For Jurkat cells, 10⁶ cells were collected by centrifugation and washed with PBS, then the pellets were resuspended in 8 M urea and 0.1 M ammonium bicarbonate (to 1 μg/μL protein). The lysates were reduced with 5 mM tris(2‐carboxyethyl)phosphine for 1 h at 37°C. Subsequently, the lysate was alkylated with 25 mM iodoacetamide for 20 min at 21°C. The lysate was diluted to 2 M urea and digested with trypsin at a ratio 1:100 (enzyme to protein) at 37°C for 15 h. The samples were spun at 20 000 × g at 4°C for 10 min. The peptides were desalted using C18 MacroSpin columns from The Nest Group according to manufacturer's instructions. After drying, the peptides were resuspended in 1% ACN and 0.1% formic acid. The Biognosys’ iRT kit, was added to all of the samples according to manufacturer's instructions (required for the DIA analysis using Biognosys’ Spectronaut).