S trap

Frozen samples (250 mg) were fractionated as above, nuclear pellets were resuspended in 50 µl of 5% SDS containing PBS and cytoplasmic fractions were mixed 1:1 with 10% SDS solution to give a final 5% SDS concentration. Proteins were digested using S-Trap (Protifi) and samples cleared of DNA/RNA by heating to 95 °C and sonication. Samples were then reduced with TCEP (5 mM final concentration, 15 min incubation at 55 °C) and alkylated with Iodoacetamide (10 mM final concentration, 10 min incubation at RT). Resulting samples were acidified with 12% Phosphoric acid (final concentration of 2.5%, v/v), followed by addition of 6 vol. of loading buffer (90% methanol, 100 mM TEAB, pH 8) and loaded onto S-Trap cartridges. Cartridges were spun at 4000×g for 30 s and washed with 90% loading buffer × 3 and flow-through discarded. Retained proteins were trypsin digested (10:1 protein: trypsin), in 50 mM TEAB, pH8.5 for 3 h at 47 °C.
Peptides were eluted, first, with 50 µl of 50 mM TEAB, followed by 50 µl of 0.2% formic acid and finally with 50 µl of 50% acetonitrile and 0.2% formic acid.

HT-29 cells were plated in 10-well plates (120,000 cells/well) in 10 ml of regular growth medium. 2 h later cells were treated with either DMSO or MS023 (5 µM). After 5 days cells were trypsinazed, washed with PBS, and pellets were stored in − 80 °C until processing. Frozen HT-29 cell pellets were lysed in 5% Sodium dodecyl sulfate (SDS, #L3771) in 50 mM Tris pH 7.4 and 100 µg of total protein were taken for the tryptic digest. The samples volume was adjusted to 50 µl with 50 mM ammonium bicarbonate, digested with trypsin using S-trap (Protifi, Huntington NY, USA)^{60 (link)} according to the manufacturer’s instructions, vacuum dried and stored in − 80 °C prior to fractionation. All chemicals were from Sigma Aldrich, St. Louis MO, USA, unless stated otherwise.

All chemicals and reagents were purchased from Sigma Aldrich (St. Louis, Michigan, USA) unless otherwise stated. For sample homogenization, two types of micropestles were acquired, one from Sigma Aldrich (#BAF199230001) and one Optima Inc., Glencoe, IL, USA, (#320302). Filter-aided sample preparation (FASP) [37 (link),38 (link)] was conducted on Microcon^® Centrifugal Filters (30 kDa molecular cut-off, Merck KGaA #MRCF0R030, purchased through Sigma Aldrich. For bead-based sample preparation, ferromagnetic beads with MagReSyn^® Amine functional groups (ReSyn Biosciences, Gauteng, South Africa) were used. For sample preparation using suspension trapping, S-Trap [51 (link)] micro-cartridges with a binding capacity of <100 µg total protein were purchased from ProtiFi (Farmingdale, NY, USA, #CO2-micro-80).
The total protein concentration was determined using a reducing-agent-compatible Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA, #23250) following the manufacturer’s instructions.

The samples were stored at −80 °C until preparation was initiated. By the beginning of the preparation, the samples were thawed and Schirmer strips incubated for 20 min in lysis buffer (5% SDS, 50 mM triethylammonium bicarbonate [TEAB]). Samples were prepared according to the S-Trap™ Micro spin column digestion protocol from ProtiFi (ProtiFi, Huntington, NY, USA) as previously described [8 (link)].
The reduction in disulphide bonds, alkylation of cysteines, and digestion in S-Trap micro columns were performed as described in a recent article [8 (link)]. The elution of peptides, recovery of hydrophobic peptides, and measurement of peptide concentration were performed as previously described [8 (link),30 (link)]. Each sample was dried in a vacuum centrifuge and stored at −80 °C until further use.

All chemicals used for preparation of nanoflow liquid chromatography–tandem mass spectrometry (nLC–MS/MS) samples were of sequencing grade and purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise stated. The Rab11A IP eluate was separated by SDS–PAGE using NuPAGE 1DE System (NuPAGE Novex 4–12% bis–tris 1.5-mm gels, Thermo Fisher Scientific, Waltham, MA). Visualization of separated proteins was performed by overnight staining with Coomassie blue G-250 solution (Thermo Fisher Scientific, Waltham, MA). The in-gel tryptic digestion followed by peptide extraction from the gel bands was performed according to previously published protocols^{67 (link)}. The extracted peptides were desalted using Poros Oligo R3 RP (PerSeptive Biosystems, Framingham, MA) P200 columns with C18 3 M plug (3 M Bioanalytical Technologies, St. Paul, MN) prior to nLC–MS/MS analysis. The Rab11A IP input samples were processed using the suspension trap (S-Trap, Protifi, Huntington, NY)^{68 (link)} mini spin column digestion protocol with minor modifications. The Rab11A IP input samples were first mixed with 20% SDS to the final concentration of 5% SDS following the manufacturer’s procedure. The peptide solution was pooled, lyophilized, and desalted prior to nLC–MS/MS.

For TMT-based whole cell proteomic analysis of primary human CD4+ T cells, resting or activated cells were washed with ice-cold PBS with Ca/Mg pH 7.4 (Sigma) and frozen at −80°C. Samples were lysed, reduced, alkylated, digested and labelled with TMT reagents (Thermo Scientific) using either iST-NHS (PreOmics GmbH; time course and single time point experiments) or S-Trap (Protifi; SBP-ΔLNGFR control experiment) sample preparation kits, according to the manufacturers’ instructions. Typically, 5e6 resting or 1e6 activated cells were used for each condition.