PXD016963

PXD016963 is an original dataset announced via ProteomeXchange.

Title	Fast and highly efficient affinity enrichment of Azide-A-DSBSO cross-linked peptides.
Description	Cross-linking mass spectrometry is an increasingly used, powerful technique to study protein-protein interactions or to provide structural information. Due to sub-stochiometric reaction efficiencies, cross-linked peptides are usually low abundant. This results in challenging data evaluation and the need for an effective enrichment. Here we describe an improved, easy to implement, one-step method to enrich azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide (DSBSO) cross-linked peptides using dibenzocyclooctyne (DBCO) coupled Sepharose������ beads. We probed this method using recombinant Cas9 and E. coli ribosome. For Cas9, the number of detectable cross-links was increased from ~100 before enrichment to 580 cross-links after enrichment. To mimic a cellular lysate, E. coli ribosome was spiked into a tryptic HEK background at a ratio of 1:2 ��������� 1:100. The number of detectable unique cross-links maintained high at ~100. The estimated enrichment efficiency was improved by factor 4 -5 (based on XL numbers) compared to enrichment via biotin and streptavidin. We were still able to detect cross-links from 0.25 ������g cross-linked E. coli ribosome in a background of 100 ������g tryptic HEK peptides, indicating a high enrichment sensitivity. In contrast to conventional enrichment techniques, like SEC, the time needed for preparation and MS measurement is significantly reduced. This robust, fast and selective enrichment method for azide-tagged linkers will contribute to map protein-protein interactions, investigate protein architectures in more depth and help to understand complex biological processes.
HostingRepository	PRIDE
AnnounceDate	2020-11-06
AnnouncementXML	Submission_2020-11-06_08:50:47.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Manuel Matzinger
SpeciesList	scientific name: Escherichia coli; NCBI TaxID: 562; scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Fusion Lumos; Q Exactive HF

Revision	Datetime	Status	ChangeLog Entry
0	2020-01-06 05:19:58	ID requested
1	2020-05-11 07:53:23	announced
⏵ 2	2020-11-06 08:50:48	announced	2020-11-06: Updated project metadata.

Matzinger M, Kandioller W, Doppler P, Heiss EH, Mechtler K, Fast and Highly Efficient Affinity Enrichment of Azide-A-DSBSO Cross-Linked Peptides. J Proteome Res, 19(5):2071-2079(2020) [pubmed]

ProteomeXchange project tag: EPIC-XS

submitter keyword: Cross-linking, mass spectrometry, DSBSO, DBCO, click reaction, affinity enrichment

Karl Mechtler
contact affiliation	IMP, Campus Vienna Biocenter 1, 1030 Vienna, Austria
contact email	karl.mechtler@imp.ac.at
lab head
Manuel Matzinger
contact affiliation	IMP / University of Vienna
contact email	manuel.matzinger@imp.ac.at
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD016963
     1. Label: PRIDE project
     2. Name: Fast and highly efficient affinity enrichment of Azide-A-DSBSO cross-linked peptides.