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Reliable identification of protein-protein interactions by crosslinking mass spectrometry.

ABSTRACT: Protein-protein interactions govern most cellular pathways and processes, and multiple technologies have emerged to systematically map them. Assessing the error of interaction networks has been a challenge. Crosslinking mass spectrometry is currently widening its scope from structural analyses of purified multi-protein complexes towards systems-wide analyses of protein-protein interactions (PPIs). Using a carefully controlled large-scale analysis of Escherichia coli cell lysate, we demonstrate that false-discovery rates (FDR) for PPIs identified by crosslinking mass spectrometry can be reliably estimated. We present an interaction network comprising 590 PPIs at 1% decoy-based PPI-FDR. The structural information included in this network localises the binding site of the hitherto uncharacterised protein YacL to near the DNA exit tunnel on the RNA polymerase.

SUBMITTER: Lenz S

PROVIDER: S-EPMC8196013 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Crosslink mass spectrometry datasets on sulfo-SDA-crosslinked affinity-purified RNA polymerase binders from SPA-pulldowns in E. coli K12, on rpoB-SPA / nusG-SPA / yacL-SPA. Cleared E. coli lysate was added to anti-FLAG M2 beads to isolate binders to rpoB (RNA polymerase), nusG or yacL (RNA polymerase binders). Elution was accomplished via TEV protease cleavage. pulldown eluate fractions were split. The heterobifunctional photoactivatable crosslinker sulfo-SDA (sulfosuccinimidyl 4,4′-azipentanoate) (Thermo Scientific Pierce, Waltham, MA, USA) was dissolved in modified lysis buffer (50 mM Hepes pH 7.2 at RT, 50 mM KCl, 10 mM NaCl, 1.5 mM MgCl2, 5% (v/v) glycerol) and immediately added to the samples at 50, 100, 250, 500 and 1000 µM. The crosslinking reaction proceeded in the dark for 2 h on ice. UV-crosslinking was achieved by irradiation with a UV laser at 365 nm for 15 seconds. Samples were frozen and stored at -20°C. Next, the samples were denatured by adding solid urea to give an 8 M solution, reduced using DTT at 10 mM following incubation at RT for 30 min and derivatized at 30 mM IAA over 20 min at RT and in the dark. LysC protease was added (protease:protein ratio ca. 1:100 (m/m)) and the samples digested for 4 h at 37°C. Then, the samples were diluted 1:5 with 100 mM ABC and trypsin was added at a ratio of approx. 1:50 (m/m). Digestion progressed for 16 h at 37 °C until stopping with TFA. Digests were cleaned up using C18 StageTips. Eluted peptides were fractionated using a Superdex Peptide 3.2/300 column (GE Healthcare) at a flow rate of 10 µl min−1 using 30% (v/v) acetonitrile and 0.1 % (v/v) trifluoroacetic acid as mobile phase(Leitner et al. 2012). Early 50 µl-fractions were collected, dried and stored at -20°C prior to LC-MS analysis. Each peptide fraction was acquired by LC-MS on a Fusion Lumos Tribrid mass spectrometer connected to an Ultimate 3000 RSLCnano system (Dionex, Thermo Fisher Scientific, Germany). Samples were resuspended in 1.6% acetonitrile 0.1% formic acid and injected onto an EASY-Spray column of 50 cm length (Thermo Scientific) running at 300 nl/min. Gradient elution using water with 0.1% formic acid and 80% acetonitrile with 0.1% formic acid was accomplished using optimised gradients for each SEC fraction (from 2% mobile phase B to 52.5% over 90 min, followed by a linear increase to 55% and 95% over 2.5 min each). Each fraction was analysed in duplicates. The settings of the mass spectrometer were as follows: Data-dependent mode with 2.5s-Top-speed setting; MS1 scan in Orbitrap at 120,000 resolution over 400 to 1,500 m/z; MS2-scan trigger only on precursors with z = 3-7+; fragmentation by HCD employing a decision tree logic with optimised collision energies; MS2 scan in Orbitrap at resolution of 60,000; dynamic exclusion was enabled upon single observation for 60 seconds. A recalibration of the precursor m/z was conducted based on high-confidence linear peptide identifications. The re-calibrated peak lists were searched against the sequences of proteins identified in a given pulldown along with their reversed sequences (as decoys) using xiSEARCH (v.1.7.6.2) for identification. MS-cleavability of the sulfo-SDA crosslinker was considered. Final crosslink lists were compiled using the identified candidates filtered to 2% FDR on residue pair-level and 5% on PPI-level with xiFDR v.2.1.5.

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Reliable identification of protein-protein interactions by crosslinking mass spectrometry.

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