PXD044316

PXD044316 is an original dataset announced via ProteomeXchange.

Title	Detection, isolation and characterization of phage-host complexes using BONCAT and click chemistry
Description	Phages are viruses that infect prokaryotes and can shape microbial communities by lysis, thus offering applications in various fields. However, challenges exist in sampling, isolation and accurate prediction of the host specificity of phages as well as in the identification of newly replicated virions in response to environmental challenges. A new workflow using biorthogonal non-canonical amino acid tagging (BONCAT) and click chemistry (CC) allowed combined analysis of phages and their hosts, the identification of newly replicated virions, and the specific tagging of phages with biotin for affinity chromatography. Replication of phage λ in Escherichia coli was selected as a model for workflow development. Specific labeling of phage λ proteins with the non-canonical amino acid 4-azido-L-homoalanine (AHA) during phage development in E. coli was confirmed by LC–MS/MS. Subsequent tagging of AHA with fluorescent dyes via CC allowed the visualization of phages adsorbed to the cell surface by fluorescence microscopy. Flow cytometry enabled the automated detection of these fluorescent phage-host complexes. Alternatively, AHA-labeled phages were tagged with biotin for purification by affinity chromatography. Despite biotinylation the tagged phages could be purified and were infectious after purification. Applying this approach to environmental samples would enable host screening without cultivation. A flexible and powerful workflow for the detection and enrichment of phages and their hosts in pure cultures has been established. The developed method lays the groundwork for future workflows that could enable the isolation of phage-host complexes from diverse complex microbial communities using fluorescence-activated cell sorting or biotin purification. The ability to expand and customize the workflow through the growing range of compounds for CC offers the potential to develop a versatile toolbox in phage research. This work provides a starting point for these further studies by providing a comprehensive standard operating procedure.
HostingRepository	PRIDE
AnnounceDate	2024-08-29
AnnouncementXML	Submission_2024-08-29_04:50:58.635.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD044316
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Patrick Hellwig
SpeciesList	scientific name: Escherichia phage Lambda; NCBI TaxID: 2681611;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Bruker Daltonics timsTOF series

Revision	Datetime	Status	ChangeLog Entry
0	2023-08-03 02:48:48	ID requested
⏵ 1	2024-08-29 04:50:59	announced

10.6019/PXD044316;

10.3389/FMICB.2024.1434301;

submitter keyword: phage, fluorescence, proteomics,BONCAT labeling, host range, sequencing, click chemistry, biotin, LC-MS/MS

Dirk Benndorf
contact affiliation	Hochschule Anhalt Fachbereich 7 - Angewandte Biowissenschaften und Prozesstechnik (BWP) Bernburger Straße 55 06366 Köthen Germany
contact email	dirk.benndorf@hs-anhalt.de
lab head
Patrick Hellwig
contact affiliation	Otto-von-Guericke-University Universitätsplatz 2 39106 Magdeburg Germany
contact email	hellwig@mpi-magdeburg.mpg.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/2024/08/PXD044316

PRIDE project URI

• PRIDE
  1. PXD044316
     1. Label: PRIDE project
     2. Name: Detection, isolation and characterization of phage-host complexes using BONCAT and click chemistry