PXD031114-1

PXD031114 is an original dataset announced via ProteomeXchange.

Title	Optimized TMT-based quantitative cross-linking mass spectrometry strategy for large-scale interactomic studies
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.
HostingRepository	PRIDE
AnnounceDate	2022-04-07
AnnouncementXML	Submission_2022-04-07_08:28:32.846.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Max Ruwolt
SpeciesList	scientific name: Escherichia coli; NCBI TaxID: 562; scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	TMT6plex-126 reporter+balance reagent acylated residue; acetylated residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2022-01-20 09:17:10	ID requested
⏵ 1	2022-04-07 08:28:33	announced

Dataset with its publication pending

submitter keyword: cross-linking, XL-MS, mass spectrometry, Lumos, FAIMS, DSSO, stepped HCD, E. coli, HEK, interference, ratio distortion, SPS, interactome

Fan Liu
contact affiliation	Leibniz-Forschungsinstitut fuer Molekulare Pharmakologie, Berlin, Germany
contact email	FLiu@fmp-berlin.de
lab head
Max Ruwolt
contact affiliation	Leibniz-Forschungsinstitut for Molecular Pharmacology
contact email	ruwolt@fmp-berlin.de
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2022/04/PXD031114

PRIDE project URI

• PRIDE
  1. PXD031114
     1. Label: PRIDE project
     2. Name: Optimized TMT-based quantitative cross-linking mass spectrometry strategy for large-scale interactomic studies