DOI: 10.5281/zenodo.15576411
title: Condensation-dependent TNS1 proximity biotinylation proteomics

description: The U2OS cell lines stably expressing Myc-BioID only, and with low and high expression of TNS1-Myc-BioID were seeded on two 10 cm plates and supplemented with biotin to a final concentration of 50 μM for at least 18 h. After labelling, the cells were washed twice with PBS and harvested into lysis buffer containing 50 mM Tris (pH 7.4), 500 mM NaCl, 0.2% SDS, 1 mM DTT and protease inhibitors (Roche). Lysates were transferred to a tube containing 20% TritonX‑100 (final concentration 2%), passed four times through a 21G needle and 50 mM Tris pH 7.4 (360 μl per plate) was added to the tubes. Subsequently, the lysates were passed additional four times through a 27G needle and centrifuged at 16000×g for 10 min at 4°C. Biotinylated proteins were captured from the cleared lysates by overnight rotation with magnetic Streptavidin beads (ReSyn Biosciences). After incubation, the beads were washed (5 min at RT per wash) twice with 2% SDS, once with a buffer containing 0.1% sodium deoxycholate, 1% Triton X-100, 500 mM NaCl, 1 mM EDTA and 50 mM HEPES (pH 7.5), and once with buffer containing 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA and 10 mM Tris.Cl (pH 7.4). Proteins were eluted in 2× reducing sample buffer saturated with biotin, heated at 70°C for 10 min and subjected to SDS-PAGE, followed by reduction, alkylation and in-gel digestion. Samples from four independent biological replicates were analysed by MS. 

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 60 min non-linear gradient was used to elute peptides (50 min from 5% to 21% solvent B followed by 22 min from 21 % to 36 min solvent B and in 5 min from 36% to 100% of solvent B, followed by 5 min wash stage with solvent B). MS data were acquired automatically using Thermo Xcalibur 4.1 software (Thermo Fisher Scientific). A data dependent acquisition method consisted of repeated cycles of one MS1 scan covering a range of m/z 300 – 1750 plus a series of HCD fragment ion scans (MS2 scans) for the most intense peptide ions from the MS1 scan. Raw data files were analysed using MaxQuant software (version 2.0.3.0) and the MS/MS spectra were searched against a human database (Uniprot-SwissProt, version 230427) using Andromeda search engine. Search parameters were set to trypsin digestion with two missed cleavages, carbamidomethyl (C) as a fixed modification, and oxidation (M), acetyl (protein N-term), biotinylation (K) and biotinylation (protein N‑term) as variable modifications. The identified peptides were filtered based on FDR 0.01. Unique peptide counts identified for each protein group were subjected to statistical analysis using SAINTexpress (Significance Analysis of INTeractome) in SPC mode with default settings (n-iter 4000, lowMode off, minFold on, normalize on).