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Reanalysis of Cross-Linking Mass Spectrometry data from a ribisomal cellular fraction for benchmarking purposes

ABSTRACT: Reanalysis of a cross-linked ribosomal cellular fraction dataset, that is a subset of a fragmentation parameters optimization dataset (PRIDE project PXD006131). The HCD fragmented part of the dataset was searched with the Cross-Linking MS identification tools OpenPepXL, pLink2, Kojak and XiSearch.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Eugen Netz

LAB HEAD: Oliver Kohlbacher

PROVIDER: PXD014520 | Pride | 2020-10-20

REPOSITORIES: Pride

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OpenPepXL: An Open-Source Tool for Sensitive Identification of Cross-Linked Peptides in XL-MS.

Netz Eugen E Dijkstra Tjeerd M H TMH Sachsenberg Timo T Zimmermann Lukas L Walzer Mathias M Monecke Thomas T Ficner Ralf R Dybkov Olexandr O Urlaub Henning H Kohlbacher Oliver O

Molecular & cellular proteomics : MCP 20201016 12

Cross-linking MS (XL-MS) has been recognized as an effective source of information about protein structures and interactions. In contrast to regular peptide identification, XL-MS has to deal with a quadratic search space, where peptides from every protein could potentially be cross-linked to any other protein. To cope with this search space, most tools apply different heuristics for search space reduction. We introduce a new open-source XL-MS database search algorithm, OpenPepXL, which offers in ...[more]

PMID: 33067342

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Comparison of OpenPepXL and xQuest on a Labeled Cross-Linking MS dataset

Project description:These data sets are part of a large study aimed at the comparison of experimental and computational workflows used in chemical cross-linking coupled to mass spectrometry. Bovine serum albumin (BSA) was used as a model protein. Two different cross-linking chemistries were used: disuccinimidyl suberate (DSS) and a combination of pimelic acid dihydrazide (PDH) and the coupling reagent, DMTMM. See related publication: Iacobucci et al., First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study, Analytical Chemistry, 91 (2019) 6953-6961, DOI: 10.1021/acs.analchem.9b00658 This project is a reanalysis of these datasets with the two Labeled Cross-Linking MS identification tools OpenPepXL and xQuest.

2020-10-20 | PXD014523 | Pride

Cross-linking mass spectrometry analysis of trimeric complex of CRM1, SNP1 and RanGTP cross-linked with BS3

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2020-10-20 | PXD014359 | Pride

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2020-12-08 | PXD012127 | Pride

Mitochondrial interactome quantitation reveals structural changes in metabolic machinery in failing murine heart

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2022-04-07 | PXD031114 | Pride

Effects of a phosphomimetic mutation and nucleotide binding on the interaction of Hsp90a with co-chaperone Aha1 analyzed by chemical cross-linking with mass spectrometry

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2019-06-17 | PXD013476 | Pride

Integrative Structural Characterization of the Human Canonical and Non-canonical Cop9 Signalosome Complexes

Project description:The COP9 signalosome (CSN) is an evolutionarily conserved protein complex that functions as a deneddylase to inactivate Cullin-RING E3 ligases for controlling protein ubiquitination. CSN possesses structural flexibility that is important for its activation upon binding to a diverse array of CRLs. The canonical and non-canonical CSN complexes consist of 8 (CSN1-8) and 9 (CSN1-9) subunits, respectively. Although CSN9 is not essential for CSN assembly and function, it appears to be important in non-catalytic regulation of CRLs by CSN. Here we employed a combinatory cross-linking mass spectrometry (XL-MS) approach to generate the largest PPI maps of human CSN complexes, which significantly enhanced the precision of integrative structural modeling. The resulting integrative structures allowed us not only to elucidate architectures of both complexes, but also to assess CSN structural dynamics that was not described in the crystal structure. In addition, we have determined CSN9 docking sites and its impact on the CSN structure. While CSN9 binding did not induce global conformational changes, it triggered subunit local reorientations that may be associated with CSN9 involvement in CSN-mediated steric regulation of CRLs.

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Project description:Cross-Linking/mass spectrometry draws structural information out of covalently linked peptide pairs. When these links do not match to previous structural models they may indicate changes of protein conformation. Unfortunately, such links can also be the result of experimental errors or artefacts. Here we describe the observation of non-covalently associated peptides during liquid chromatography-mass spectrometry analysis which can easily be misidentified as cross-linked. Strikingly, they often mismatch to the protein structure or may falsely suggest protein homo-dimerization. Non-covalently associated peptides presumably form during ionization and can be distinguished from cross-linked peptides by observing co-elution of the corresponding linear peptides in MS1, as well as the presence of the individual peptide fragments in mixed MS2 spectra. Interestingly, non-covalently associated peptides seem sensitive to ionization source design as we see them more prevalently on a Q Exactive than on an Orbitrap Velos mass spectrometer. Finally we show how more disruptive ionization settings such as moderate in-source fragmentation suppresses their presence.

2019-01-25 | PXD010895 | Pride

Optimizing the parameters governing the fragmentation of cross-linked peptides in a tribrid mass spectrometer

Project description:We compared the five different ways of fragmentation available on a tribrid mass spectrometer and optimized their collision energies with regard to optimal sequence coverage of cross-linked peptides. We created a library of bis(sulfosuccinimidyl)suberate (BS3/DSS) cross-linked precursors, derived from the tryptic digests of three model proteins (Human Serum Albumin, creatine kinase and myoglobin). This enabled in-depth targeted analysis of the fragmentation behavior of 1065 cross-linked precursors using the five fragmentation techniques: collision-induced dissociation (CID), beam-type CID (HCD), electron-transfer dissociation (ETD), and the combinations ETciD and EThcD. EThcD gave the best sequence coverage for cross-linked m/z species with high charge-density, while HCD was optimal for all others. We tested the resulting data-dependent decision tree against collision energy-optimized single methods on two samples of differing complexity (a mix of eight proteins and a highly complex ribosomal cellular fraction). For the high complexity sample the decision tree gave the highest number of identified cross-linked peptide pairs passing a 5% false discovery rate (on average ~21% more than the second best, HCD). For the medium complexity sample the higher speed of HCD proved decisive. Currently, acquisition speed plays an important role in allowing the detection of cross-linked peptides against the background of linear peptides. Enrichment of cross-linked peptides will reduce this role and favor methods that provide spectra of higher quality.

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