PXD010422

PXD010422 is an original dataset announced via ProteomeXchange.

Title	eXL-MS: An enhanced Cross-Linking Mass Spectrometry Workflow to Study Protein Complexes.
Description	The analysis of proteins and protein complexes by cross-linking mass spectrometry (XL-MS) has expanded in the last dec-ade. However, mostly used approaches suffer important limitations in term of efficiency and sensitivity. We describe here a new workflow based on the advanced use of the trifunctional cross-linker NNP9. NNP9 carries an azido group allowing the quantitative and selective introduction of a biotin molecule into cross-linked proteins. The incorporation is performed by click-chemistry using an adapted version of the enhanced filter-aided sample preparation (eFASP) protocol. This protocol, based on the use of a molecular filter, allows a very high recovery of peptides after enzymatic digestion and complete re-moval of contaminants. This in turn offers the possibility to analyze very large membrane proteins solubilized in detergent. After trypsin digestion, biotinylated peptides can be easily enriched on monoavidin beads and analyzed by LC-MS/MS. The whole workflow was developed on creatine kinase in presence of detergent. It led to a drastic improvement in the number of identified cross-linked peptides (407 vs 81), compared to the conventional approach using a gel-based separation. One great advantage of our eXL-MS workflow is its high efficiency, allowing the analysis of very low amount of material (15 mi-crograms). We also demonstrate that Higher-Energy Collision Dissociation (HCD) outperforms Electron-Transfer/Higher-Energy Collision Dissociation (EThcD) in term of number of cross-linked peptides identified, but EThcD leads to better se-quence coverage than HCD and thus easier localization of cross-linking sites.
HostingRepository	PRIDE
AnnounceDate	2021-08-11
AnnouncementXML	Submission_2021-08-11_00:40:34.168.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Martial Rey
SpeciesList	scientific name: Oryctolagus cuniculus (Rabbit); NCBI TaxID: 9986;
ModificationList	No PTMs are included in the dataset
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2018-07-13 03:19:06	ID requested
⏵ 1	2021-08-11 00:40:34	announced

Rey M, Dupr, é M, Lopez-Neira I, Duchateau M, Chamot-Rooke J, eXL-MS: An Enhanced Cross-Linking Mass Spectrometry Workflow To Study Protein Complexes. Anal Chem, 90(18):10707-10714(2018) [pubmed]

curator keyword: Technical, Biological

submitter keyword: Creatine Kinase, cross-link, mass spectrometry, click chemistry, monoavidin biotin affinty enrichment

Julia Chamot-Rooke
contact affiliation	Mass Spectrometry for Biology Unit, CNRS USR 2000, Institut Pasteur, Paris, France.
contact email	julia.chamot-rooke@pasteur.fr
lab head
Martial Rey
contact affiliation	CNRS, Pasteur
contact email	martial.rey@pasteur.fr
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD010422
     1. Label: PRIDE project
     2. Name: eXL-MS: An enhanced Cross-Linking Mass Spectrometry Workflow to Study Protein Complexes.