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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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Dataset's files

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			Action	DRS
	B160812_02_Lumos_LK_IN_190_Francis_Rib_Fra_HCD_DT_1.mgf	Mgf
	B160812_02_Lumos_LK_IN_190_Francis_Rib_Fra_HCD_DT_1.mzML	Mzml
	B160812_02_Lumos_LK_IN_190_Francis_Rib_Fra_HCD_DT_1.mzid	Mzid
	B160812_02_Lumos_LK_IN_190_Francis_Rib_Fra_HCD_DT_1.raw	Raw
	B160812_07_Lumos_LK_IN_190_Francis_Rib_Fra_HCD_DT_2.mgf	Mgf

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Publications

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

Project description:An X-ray crystallography structure of the trimeric nuclear export complex of CRM1, SNP1 and RanGTP is available on the PDB database with the ID 3GJX. To get an XL-MS dataset of a clean sample of a stable protein complex for benchmarking purposes, another sample from the protein expression, purification and complex assembly employed in the crystallography study was also cross-linked and analyzed by mass spectrometry. The tools OpenPepXL, pLink 2, Kojak, StavroX, XiSearch and xQuest were used to analyze this dataset with comparable search settings.

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Reanalysis of a synthetic cross-linked peptide library with OpenPepXL for benchmarking purposes

Project description:Reanalysis of a synthetic crosslinked peptide library dataset. The three replicates of the XL-MS experiment with the non-cleavable cross-linker DSS from the dataset published in Beveridge et al., Nature Commun., 2020 (PRIDE project PXD014337) were searched with the XL-MS identification tool OpenPepXL for benchmarking purposes.

2020-10-20 | PXD021417 | Pride

TEAD1-DBD-DNA interaction studied by structural MS

Project description:TEAD transcription factors mediate gene expression regulation through interactions with their DNA response M-CAT motif. They are active mainly during growth and development and induce gene expression of proteins involved in cell proliferation, differentiation or apoptosis prevention. TEADs and many of their target proteins are known to be upregulated in several types of cancers. To study the interaction of DNA binding domain of TEAD1 with M-CAT motifs of different orientations originating from regulatory regions of human genes (CTGF, SRF, C-MYC and GLUT1), dissociation constant of each complex was determined using fluorescence anisotropy assay. Structural mass spectrometry techniques (H/D exchange and quantitative chemical cross-linking) and molecular docking were utilized for structural characterization of the binding interface. In-vitro results were correlated with situation in living cells studied by ChIP and Q-PCR.

2020-12-08 | PXD012127 | Pride

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

Project description:Advancements of cross-linking mass spectrometry (XL-MS) for structural analysis of proteins bridges the gap between purified systems and native tissue environments. Here, we utilize isobaric quantitative protein interaction reporter technology (iqPIR) to further extend XL-MS to the first system-wide comparative study of mitochondrial proteins from healthy and diseased murine hearts. The failing heart interactome includes 544 statistically significant cross-linked peptide pairs altered in disease condition. Structural insight into ketone oxidation metabolons, OXPHOS machinery, and nucleotide transporter hybrid-conformations, support mitochondrial remodeling in failing heart while bringing forth new hypotheses for pathological mechanisms. Application of quantitative cross-linking technology in live tissue provides molecular-level insight to complex biological systems difficult to model in cell culture, thus providing a valuable resource for study of human diseases.

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Optimized TMT-based quantitative cross-linking mass spectrometry strategy for large-scale interactomic studies

Project description:Cross-linking mass spectrometry is a powerful method for the investigation of protein-protein interactions from highly complex samples. XL-MS combined with tandem mass tag labeling holds the promise of large-scale PPI quantification. However, a robust and efficient TMT-based XL-MS quantification method has not yet been established due to the lack of a benchmarking dataset and thorough evaluation of various MS parameters. To tackle these limitations, we generate a two-interactome dataset by spiking-in TMT-labeled cross-linked E. coli lysate into TMT-labeled cross-linked HEK293T lysate using a defined mixing scheme. Using this benchmarking dataset, we assess the efficacy of cross-link identification and accuracy of cross-link quantification using different MS acquisition strategies. For identification, we compare various MS2- and MS3-based XL-MS methods, and optimize stepped HCD energies for TMT-labeled cross-links. We observed a need for notably higher fragmentation energies compared to unlabeled cross-links. For quantification, we assess the quantification accuracy and dispersion of MS2-, MS3- and synchronous precursor selection-MS3-based methods. We show that a stepped HCD-MS2 method with stepped collision energies 36-42-48 provides a vast number of quantifiable cross-links with high quantification accuracy. This widely applicable method paves the way for multiplexed quantitative PPI characterization from complex biological systems.

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

Project description:Complex conformational dynamics are essential for the chaperone function of heat shock protein 90 (Hsp90), including transient, ATP-biased N-domain dimerization establishing ATPase competence. Biochemical data demonstrate that the intrinsic, but weak, ATP hydrolyzing activity of Hsp90 is markedly enhanced by the co-chaperone Aha1. However, cellular concentration of Aha1 is substoichiometric relative to Hsp90. In cells, interaction of this important co-chaperone with Hsp90 is up-regulated by posttranslational modifications (PTMs), including phosphorylation of a highly conserved tyrosine (Y313 in Hsp90a). Here we use chemical cross-linking with mass spectrometry to explore the the impacts of a phosphomimetic mutation (Y313E) and binding of a non-hydrolyzable ATP analog (AMP-PNP),on the structure Hsp90a and its interaction with Aha1.

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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Non-covalently associated peptides are observed during liquid chromatography-mass spectrometry and affect cross-link analyses

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.

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