DOI: 10.5281/zenodo.18405274
title: Yeast RNAPII complex streptavidin pull-down in presence of arsenic-biotin, analyzed by bottom-up LC-MS

description: RNAPII complex purification

Approximately 10¹¹ logarithmically growing untagged or Rpb1-HTP (His₆–TEV cleavage site–2× protein A) tagged cells were harvested by centrifugation, extensively washed with water and YEB buffer (25 mM HEPES–KOH pH 7.9, 100 mM KCl, 2 mM MgCl₂, 5% glycerol, 0.1% Triton X-100, 1 mM DTT, protease inhibitors [Sigma-Aldrich], 1 mM PMSF), and finally resuspended in 4 ml YEB. Cells were lysed in a bead beater at 4°C, and whole-cell extracts were centrifuged for 15 min at 15,000 rpm. The resulting protein extracts were incubated with 2 kU/ml benzonase and 100 µg/ml RNase A for 1 h at 4°C. The extracts were then incubated with IgG Sepharose beads (Cytiva) for 3 h at 4°C with rotation. Next, the beads were washed several times with IPP buffer (10 mM Tris pH 7.9, 100 mM NaCl, 0.1% Triton X-100) and then with TEV buffer (50 mM Tris pH 7.9, 0.2 mM EDTA, 100 mM NaCl, 0.1% Triton X-100, 1 mM DTT). To release Rpb1, the IgG Sepharose beads were incubated overnight at 4°C in TEV buffer containing 400 U/ml TEV protease. The next day, the beads were separated from the buffer, and the resulting eluate was incubated with 200 µl Ni-NTA resin for 1 h at 4°C with rotation. The beads were washed three times with Ni buffer (20 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole, pH 7.4), followed by protein elution by 5 min incubation at room temperature in Ni buffer conatinig 500 mM imidazole. The resulting eluate was loaded onto Zeba™ Spin Desalting Columns. The purified RNAPII complex was suspended in IP buffer (20 mM sodium phosphate, 300 mM NaCl, pH 7.4. The RNAPII-containing solution was then divided into two aliquots. One aliquot was supplemented with 25 µM biotin-conjugated arsenic (As-biotin), whereas the other was left untreated. As-biotin was also added to the sample prepared from the untagged strain. All samples were incubated for 30 min at room temperature.

In parallel, 30 µl streptavidin magnetic beads were blocked for 20 min at 4°C in IP buffer containing 0.2% BSA, then extensively washed with IP buffer. After the 30-min incubation, 10 µl of the blocked streptavidin beads were added to each sample and incubated for 1 h at 4°C with occasional mixing. Lysates were then removed, the beads were washed twice with IP buffer and twice with IP buffer containing 0.25% NP-40 and 500 mM NaCl, washed again with IP buffer, and finally resuspended in IP buffer.

Bottom-up LC-MS analysis of the RNAPII As-biotin pull-down

Sodium deoxycholate was added to a final concentration of 1% to bead samples, which were then incubated at 65°C for 10 minutes. Subsequently, 300 ng of trypsin was added to the sample for an overnight on-bead digestion in 37°C. Next day the solution was separated from the beads, acidified and sodium deoxycholate was removed by centrifugation. The supernatant was then desalted using a STAGE tip [1]. Obtained peptide pellet was resuspended in 0.1% formic acid (FA), 3% acetonitrile (ACN) solution.LC-MS was performed on an M-Class Acquity UPLC connected to a Synapt XS HDMS equipped with a nanoESI source. Mobile phase A consisted of H2O+0.1% FA, while mobile phase B of ACN+0.1% FA. An 8–40%B 24min linear gradient at a 300nL/min flow rate was applied for sample separation on a C18 BEH 75μmx250mm analytical column kept at 65°C. A 3-minute sample trapping step was performed prior to analytical separation. Data were collected in ESI+ using DDA mode, with MS and MS/MS scan rates of 0.2s in 250–1250 (MS) and 100–1800 (MS/MS) m/z ranges. Analyser mode was set to Resolution. The top 6 precursors from each MS scan with charges 2+, 3+, and 4+ were selected for MS/MS with 1 scan allowed per transition. Collision energy ramp determined specifically for each m/z (start LM, HM: 15–40 V; end LM, HM: 18–50 V) was applied on the trap cell. Source conditions were fine-tuned. A (Glu1)-Fibrinopeptide B solution was acquired in-parallel as lockmass, and correction was applied post-acquisition.Raw processing was performed using Progenesis QI for Proteomics (Non-linear Dynamics). Obtained spectra were first searched against the PRIDE Contaminants Library with following parameters: precursor tolerance=7.5 ppm; fragment tolerance=25 ppm; fragments per peptide=10; sharing hits across charge states was allowed. Next, an .mgf file was exported (excluding queries from the library search not related to yeast proteins) and searched using Mascot Server v2.8.0.1 (Matrix Science) against the Saccharomyces cerevisiae protein sequence databank (UP000000757) to which Streptavidin sequence was appended (UniProt entry). The search parameters were: peptide mass tolerance: 7.5 ppm; fragment mass tolerance: 25 ppm; max. protein mass: 1 MDa; digest enzyme: trypsin; max. missed cleavages: 2; variable modification: Oxidation of methionine, Acetyl of protein N-term; FDR (PSM): 1%. Protein hits were grouped into families. Progenesis protein output list was exported, and spectral counts for the Rbp complex subunits were analyzed across samples.

[1]     J. Rappsilber, Y. Ishihama and M. Mann, "Stop and Go Extraction Tips for Matrix-Assisted Laser Desorption/Ionization, Nanoelectrospray, and LC/MS Sample Pretreatment in Proteomics," Analitycal Chemistry, vol. 75, no. 3, p. 663–670, 2003.