DOI: 10.5281/zenodo.17408116
title: GFP-Tensin 1 phosphoproteomics upon sodium arsenite treatment in U2OS cells

description: Tensin 1 (TNS1) is a key component of integrin adhesion and plays a key role in linking the actin cytoskeleton with the ECM through interaction with integrin heterodimers. TNS1 forms functional and reversible condensates in cells in response to different extracellular stimuli that act as reservoirs of adhesion components. Here we analysed the role of phosphorylation on TNS1 condensation. Using phosphoproteomics of affinity-purified GFP-TNS1 from control and sodium arsenite-treated U2OS cells, we identify the phosphosites regulating TNS1 condensation. 

U2OS cells expressing GFP-TNS1 were seeded on one 15 cm plate and treated with 1mM sodium arsenite or control for 1h. Cells were washed twice with ice-cold PBS, scraped into residual PBS, transferred into 1.5 ml tube, and centrifuged at 400×g for 2 min at 4°C. Cell pellet was resuspended in 500 μl of lysis buffer containing 75 mM Tris (pH 7.4), 200 mM NaCl, 2 mM MgCl2, 1.5% Triton X-100 and 0.75% NP-40, supplemented with Complete protease inhibitors mix (Roche), PhosStop phosphatase inhibitors mix (Roche), and GENIUS Nuclease (Santa Cruz) in recommended concentrations. The lysates were incubated on ice for 20 min with occasional gentle vortexing to facilitate cell lysis and centrifuged at 13000×g for 10 min at 4°C. Part of the cleared lysates was heated with 8× reducing sample buffer to generate total lysate samples. The remining lysate was incubated for 2 h at 4°C with GFP-Trap Magnetic Agarose (Chromotek) previously washed twice with a wash buffer containing 50 mM Tris pH 7.4, 150 mM NaCl and 1% NP-40. After the incubation, the beads were washed once with lysis buffer, twice with wash buffer, three times with TBS (50 mM Tris pH 7.4, 150 mM NaCl) and stored dry at -80°C until further processed by on-bead digestion (Trypsin). The LC-ESI-MS/MS analyses were performed on a nanoflow HPLC system (Easy-nLC1200, Thermo Fisher Scientific) coupled to the Exploris 480 mass spectrometer (Thermo Fisher Scientific) equipped with a nano-electrospray ionization source and FAIMS interface. Digested peptide samples were dissolved in 0.1% formic acid and injected for analysis. Wash runs were submitted between samples to reduce peptide carry-over. Peptides were first loaded on a trapping column and subsequently separated inline on a 15 cm C18 column (75 μm x 15 cm, ReproSil-Pur 3 μm 120 Å C18-AQ, Dr. Maisch HPLC GmbH). The mobile phase consisted of water with 0.1% formic acid (solvent A) and acetonitrile/water (80:20 (v/v)) with 0.1% formic acid (solvent B). A 120 min gradient was used (62 min from 5 % to 21 % solvent B followed by 48 min from 21 % to 36 % solvent B and in 5 min from 36% to 100% of solvent B, followed by 5 min wash stage with solvent B). Samples were analysed by a data independent acquisition (DIA) LC-MS/MS method. MS data were acquired automatically using Thermo Xcalibur 4.6 software (Thermo Fisher Scientific). In the DIA method, a duty cycle contained a full scan (m/z 395 – m/z 1005) and 30 DIA MS/MS scans covering the m/z range of 400 -1000 with variable width isolation windows. 

Raw data from 4 independent replicates were analysed in Spectronaut (Biognosys, version 19.2.). DirectDIA approach was used to identify proteins against human protein database (Uniprot-SwissProt, version 2409) and label-free quantifications were performed using MaxLFQ. Fixed modifications were set to carbamidomethyl (C), variable modifications were set to oxidation (M), acetyl (protein N-term) and phospho (STY). The identified peptides were filtered based on FDR 0.01 and phosphopeptides were filtered based on localisation probability set to 0.7. Normalisation on GFP-TNS1 quantity was performed and the differential abundance of the identified phosphosites was tested using unpaired t-test.