PXD017584

PXD017584 is an original dataset announced via ProteomeXchange.

Title	Protocol for high-yield production of photo-leucine-labeled proteins in Escherichia coli
Description	Cross-linking mass spectrometry (XL-MS) is an emerging technique to obtain structural information of biomacromolecules and their complexes in cells and in vitro. In particular, certain photo-reactive amino acids (pA) such as photo-leucine (pLeu) and photo-methionine can provide unique short-distance information on structural core regions of proteins. Here, we present a protocol for high-yield incorporation of pLeu in proteins recombinantly expressed in Escherichia coli. The protein is expressed at high cell densities, which reduces the required amount of the photo-reactive amino acid by a factor of 10 compared to standard protocols, while maintaining high incorporation rates. For the two chaperones Trigger Factor and SecB, up to 3 mg of photo-leucine labeled protein were thus obtained from 100 ml of cell culture, with label incorporation rates of up to 34%. For Trigger factor, UV-induced cross-linking lead to the identification of 12 cross-links that are in agreement with published three-dimensional structures. The accessibility of milligram amounts of photo-leucine-labeled proteins at low costs will be highly useful to address structural biology questions.
HostingRepository	PRIDE
AnnounceDate	2020-05-26
AnnouncementXML	Submission_2020-05-26_05:24:45.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Bastian Kohl
SpeciesList	scientific name: Escherichia coli; NCBI TaxID: 562;
ModificationList	carbamoylated residue
Instrument	Orbitrap Fusion Lumos; LTQ Orbitrap Elite

Revision	Datetime	Status	ChangeLog Entry
0	2020-02-18 05:21:57	ID requested
⏵ 1	2020-05-26 05:24:46	announced

Kohl B, Br, ü, derlin M, Ritz D, Schmidt A, Hiller S, . J Proteome Res, 19(8):3100-3108(2020) [pubmed]

submitter keyword: Photo-leucine, Mass spectrometry, high-cell density culture, UV cross-linking, Molecular chaperones

Sebastian Hiller
contact affiliation	University of Basel Biozentrum FG Sebastian Hiller Klingelbergstrasse 70 4056 Basel
contact email	sebastian.hiller@unibas.ch
lab head
Bastian Kohl
contact affiliation	University of Basel Biozentrum FG Sebastian Hiller Klingelbergstrasse 70 4056 Basel
contact email	bastian.kohl@unibas.ch
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/2020/05/PXD017584

PRIDE project URI

• PRIDE
  1. PXD017584
     1. Label: PRIDE project
     2. Name: Protocol for high-yield production of photo-leucine-labeled proteins in Escherichia coli