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.

Improved Peptide Backbone Fragmentation Is the Primary Advantage of MS-Cleavable Crosslinkers

“…Many key advances have been made in recent years to expand the complexity of the samples that can be analyzed with this technology. These include the database search software, 1 − 3 false discovery rate (FDR) estimation, 4 and the enrichment of crosslinked peptides. 5 − 7 One of the key problems when identifying crosslinked peptides is that one must, in principle, identify two peptides from the same MS1 signal.…”

Leveraging Parameter Dependencies in High-Field Asymmetric Waveform Ion-Mobility Spectrometry and Size Exclusion Chromatography for Proteome-wide Cross-Linking Mass Spectrometry

“…Aliquots of the 26S preparation, before and after buffer exchange, were processed as described earlier. 23 Cross-linking with BS3 was conducted for 2 h on ice at a protein/cross-linker ratio of 1:3.2 (w/w) (to 0.4 mg/mL BS3) until adding ABC to 50 mM (for cross-link titration preexperiment, see Figure S11 ). The sample was dried and solubilized in 8 M urea and 100 mM ABC and reduced/alkylated.…”

“…Precursors were fragmented with stepped NCEs of 18, 24, and 30%. 23 Each measurement used an internal stepping CV pair as follows: −30/–60, −35/–65, −40/–70, −45/–75, −50/–80, and −55/–85 V. A 30 V offset was used to pair two CVs. We paired CVs such that we combined one CV leading to more precursors with one that led to fewer precursors to reduce the effect of time restriction on precursor selection for comparing individual CV values.…”

“…Cross-linker modifications were defined as variable. 23 For 293T cells, variable cross-linker modifications were only considered for linear peptides. Cross-linker specificities were defined as reacting with Lys, Tyr, Ser, Thr, and the peptide N-terminus.…”

“…Loss masses were enabled to account for the DSSO linker cleavage. 23 A “noncovalent cross-linker” with zero mass was defined to flag the spectra putatively arising from gas phase-associated peptides, which were removed from the list of spectra prior to false discovery rate (FDR) estimation. 32 Search results were filtered prior to FDR estimation to cross-link spectrum matches (CSMs) with a minimum of three matched fragments per peptide (two for 26S proteasome) and a delta score ≥0.1.…”