Reliable identification of protein-protein interactions by crosslinking mass spectrometry.
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ABSTRACT:
SUBMITTER: Lenz S
PROVIDER: S-EPMC8196013 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Covalent crosslinking and mass spectrometry techniques hold great potential in the study of multiprotein complexes, but a major challenge is the inability to differentiate intra- and inter- protein crosslinks in homomeric complexes. In the current study we use CYP102A1, a well-characterized homodimeric P450, to examine a subtractive method that utilizes limited crosslinking with disuccinimidyl dibutyric urea (DSBU) and isolation of the monomer, in addition to the crosslinked dimer, to identify inter-monomer crosslinks. The utility of this approach was examined with the use of MS-cleavable crosslinker DSBU and recently published cryo-EM based structures of the CYP102A1 homodimer. Of the 31 unique crosslinks found, 26 could be fit to the reported structures whereas 5 exceeded the spatial constraints. Not only did these crosslinks validate the cryo-EM structure, they point to new conformations of CYP102A1 that bring the flavins in closer proximity to the heme.
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Project description:Modeling macromolecular assemblies with restraints from crosslinking mass spectrometry (XL-MS) tends to focus solely on distance violation. Recently, we identified three different modeling features inherent in crosslink data: (1) expected distance between crosslinked residues; (2) violation of the crosslinker's maximum bound; and (3) solvent accessibility of crosslinked residues. Here, we implement these features in a scoring function. cMNXL, and demonstrate that it outperforms the commonlyused crosslink distance violation. We compare the different methods of calculating the distance between crosslinked residues, which shows no significant change in performance when using Euclidean distance compared with the solvent-accessible surface distance. Finally, we create a combined score that incorporates information from 3D electron microscopy maps as well as crosslinking. This achieves, on average, better results than either information type alone and demonstrates the potential of integrative modeling with XL-MS and low-resolution cryoelectron microscopy.
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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.
2021-04-14 | PXD024148 | JPOST Repository
Project description:Crosslink mass spectrometry dataset on soluble, SEC-fractionated high-molecular weight proteome of E. coli, crosslinked with BS3 or DSSO.
This data was used to identify protein-protein interactions and to obtain information on protein (complex) topologies. In the end, the data was used for finding and evaluating a reliable approach for error control in crosslink mass spectrometry, particularly for PPI-studies.
Cleared E. coli lysate was fractionated in the range of 3 MDa to 150 kDa (44 fractions). After taking aliquots for quantitative proteomics (JPST000843 ), the aliquots were split and the proteins in each fraction crosslinked (with BS3 or DSSO) and pooled again for each crosslinker set. Then, the pool was precipitated, derivatized and proteolysed in-solution. Digests were fractionated by SCX and hSAX (9x10 offline-fractionation matrix). Afterward, each peptide fraction was acquired via LC-MS on Q Exactive HF. Raw data was pre-processed (denoising, peak-list generation, error correction) and this data searched with xiSEARCH 1.6.746 using differing databases. FDR was calculated using xiFDR (version 2.0dev) under various settings to probe several FDR approaches.
2021-04-14 | PXD019120 | JPOST Repository
Project description:Quantitative proteomics dataset on soluble, SEC-fractionated high-molecular weight proteome of E. coli for protein coelution analysis.
This data was used to identify proteins for a subsequent crosslinking mass spectrometry search (DB creation) and to correlate protein elution behaviour to validate/falsify potential PPIs from the CLMS-based approach.
Cleared E. coli lysate was fractionated in the range of 3 MDa to 150 kDa (44 fractions). The proteins in each fraction were precipitated, derivatized and proteolysed in-solution. Then, each fraction was acquired via LC-MS on Q Exactive HF and the raw data searched with MaxQuant with iBAQ quantitation enabled.
2021-04-14 | PXD019004 | JPOST Repository
Project description:Quantitative proteomics dataset on soluble, affinity-enriched proteins from SPA-pulldowns in E. coli K12, on rpoB-SPA / nusG-SPA / yacL-SPA.
This data was used to identify proteins for enrichment analysis and subsequent crosslinking mass spectrometry search (DB creation).
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. A mock K12 E.coli lysate ETV elution was used to assess protein enrichment in the obtained (specific) eluates.
The proteins in each fraction were precipitated using acetone, derivatized and proteolysed in-solution. Each sample was acquired as injection triplicate via LC-MS on Q Exactive HF and the raw data searched with MaxQuant with LFQ and iBAQ quantitation enabled. Further processing was done using Perseus.
2021-04-14 | PXD024146 | JPOST Repository
Project description:We present a concise workflow to enhance the mass spectrometric detection of crosslinked peptides by introducing sequential digestion and the crosslink identification software xiSEARCH. Sequential digestion enhances peptide detection by selective shortening of long tryptic peptides. We demonstrate our simple 12-fraction protocol for crosslinked multi-protein complexes and cell lysates, quantitative analysis, and high-density crosslinking, without requiring specific crosslinker features. This overall approach reveals dynamic protein-protein interaction sites, which are accessible, have fundamental functional relevance and are therefore ideally suited for the development of small molecule inhibitors.
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Project description:Crosslinking mass spectrometry is a powerful tool to study protein-protein interactions under native or near-native conditions in complex mixtures. Through novel search controls, we show how biassing results towards likely correct proteins can subtly undermine error estimation of crosslinks, with significant consequences. Without adjustments to address this issue, we have misidentified an average of 260 interspecies protein-protein interactions across 16 analyses in which we synthetically mixed data of different species, misleadingly suggesting profound biological connections that do not exist. We also demonstrate how data analysis procedures can be tested and refined to restore the integrity of the decoy-false positive relationship, a crucial element for reliably identifying protein-protein interactions.
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Project description:Neuronal nitric oxide synthase (nNOS) is a homodimeric cytochrome P450-like enzyme that catalyzes the conversion of L-arginine to nitric oxide in the presence of NADPH and molecular oxygen. The binding of calmodulin (CaM) to a linker region between the FAD/FMN-containing reductase domain, and the heme-containing oxygenase domain is needed for electron transfer reactions, reduction of the heme, and NO synthesis. Due to the dynamic nature of the reductase domain and low resolution of available full-length structures, the exact conformation of the CaM-bound active complex during heme reduction is still unresolved. Interestingly, hydrogen-deuterium exchange and mass spectrometry studies revealed interactions of the FMN domain and CaM with the oxygenase domain for iNOS, but not nNOS. This finding prompted us to utilize covalent crosslinking and mass spectrometry to clarify interactions of CaM with nNOS. Specifically, MS-cleavable bifunctional crosslinker disuccinimidyl dibutyric urea was used to identify thirteen unique crosslinks between CaM and nNOS as well as 61 crosslinks within the nNOS. The crosslinks provided evidence for CaM interaction with the oxygenase and reductase domain residues as well as interactions of the FMN domain with the oxygenase dimer. Cryo-EM studies, which gave a high-resolution model of the oxygenase domain, along with crosslink-guided docking provided a model of nNOS that brings the FMN within 15 Å of the heme in support for a more compact conformation than previously observed. These studies also point to the utility of covalent crosslinking and mass spectrometry in capturing transient dynamic conformations that may not be captured by hydrogen-deuterium exchange and mass spectrometry experiments.
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