ZAR1-EYFP, ZAR1delZF-EYFP vs. EYFP-empty were overexpressed in HEK293T cells (24 h), and pulldown was performed according to manufacturer's protocol by GFP Trap (ChromoTek). Triplicate sample pairs were processed by off-bead digest, strong anion exchange (SAX) extraction, and dimethyl-labelling, followed by LC-MS2. I brief, beads were resuspended in two volumes urea buffer (6 M urea, 2 M thiourea, 10 mM dithiothreitol, 10 mM HEPES, pH 8.0) and incubated shaking for 30 min at room temperature. Cysteins were alcylated at 55 mM final concentration of iodoacetamide, shaking at room temperature and in the dark for another 30 min. Peptidolysis was then initiated with 0.5 μg Lys-C (Wako Chemicals GmbH) for 3 h shaking at room temperature, followed by dilution to 2 M urea/thiourea, addition of 0.5 μg trypsin (Serva) and an overnight shaking incubation at room temperature. Peptide-containing supernatants were brought to 1% NH3 and loaded onto three-layer SAX tips equilibrated previously with 30 μl of 0.1% NH3. After sequential washes with 30 μl 0.1% NH3 and 30 μl NH3 in 2-propanol, respectively, columns were syringe dried, peptides eluted using 30 μl 80% acetonitrile, 0.1% formic acid and vacuum dried. In-solution chemical labelling was performed as described [71, 72]. Peptides were resolubilized and acidified using a final concentration of 0.1% TFA. Free amines were differentially modified by reductive dimethylation. The labelling reaction was quenched on ice using ammonia solution and formic acid. Differentially labelled samples were mixed 1:1 by volumes and desalted on oligo R3 columns. The subsequent LC-MS2 analysis used an in-house packed 70 μm ID, 15 cm reverse phase column emitter (ReproSil-Pur 120 C18-AQ, 1.9 μm, Dr. Maisch GmbH) with a buffer system comprising solvent A (5% acetonitrile, 1% formic acid) and solvent B (80% acetonitrile, 1% formic acid). Relevant instrumentation parameters are extracted using MARMoSET [73] and included in the supplementary material. Peptide/protein group identification & quantitation was performed using the MaxQuant suite of algorithms [74, 75] (v. 1.6.3.4) against the human uniprot database (canonical and isoforms; downloaded on 2019/01/23; 169,389 entries) using the parameters documented in the supplementary material.
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