PXD031114

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

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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