PXD005262

PXD005262 is an original dataset announced via ProteomeXchange.

Title	A protocol for global quantification of phosphoproteins combining metabolic labeling and gel-based proteomics
Description	Differential proteomics targeting the abundance level and changes of proteins is commonly used in biology to determine changes induced by different physiological states in living organisms. Here, current methods comprise both label-free and label-based proteomics approaches. More specifically, besides the experimental view on changes on protein abundances, differences in the level of post translational modifications (PTM) like phosphorylations are of interest in physiological studies due to the anticipated role of PTM in regulatory processes. Here, particularly in microbes, both identification and quantification of phosphorylated proteins is hampered by their low abundance. They can be quantified by comparison of spot intensities on 2D gels after functional phosphoprotein staining or by stable isotope labeling combined with phosphopeptide enrichment. To combine the best of two worlds, we combined 14N/15N metabolic labeling with preceding protein separation on 2D gels and visualization of phosphorylations by functional staining in a proof-of-principle experiment. In our workflow, a global 15N heavy standard spiked in light, 14N based cell extracts was used to allow for relative quantification of changes in phosphorylation levels in Bacillus pumilus exposed to hydrogen peroxide stress comparing control and stress conditions. Mapping of putative protein phosphorylation events on the protein level was followed by protein identification and determination of molecular phosphosites by LC-MS/MS. Altogether, we were able to map 19 putatively phosphorylated proteins and could calculate the quantitative changes of stressed vs. unstressed cells for phosphorylated and non- phosphorylated peptides for 12 of these proteins to demonstrate the practicability of this method.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_04:33:49.960.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Andreas Otto
SpeciesList	scientific name: Bacillus pumilus; NCBI TaxID: 1408;
ModificationList	phosphorylated residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	LTQ Orbitrap Velos

Revision	Datetime	Status	ChangeLog Entry
0	2016-11-03 00:54:47	ID requested
1	2017-10-02 04:35:10	announced
⏵ 2	2024-10-22 04:33:52	announced	2024-10-22: Updated project metadata.

Hentschker C, Dewald C, Otto A, B, ü, ttner K, Hecker M, Becher D, Global quantification of phosphoproteins combining metabolic labeling and gel-based proteomics in B. pumilus. Electrophoresis, 39(2):334-343(2018) [pubmed]

10.1002/elps.201700220;

curator keyword: Technical

submitter keyword: peroxide stress adaptation,gel-based proteomics, phosphorylation, 14N/15N metabolic labeling, Bacillus pumilus, quantification

Dörte Becher
contact affiliation	Dörte Becher Department of Microbial Proteomics, Institute for Microbiology, Ernst-Moritz-Arndt-University Greifswald, Friedrich-Ludwig-Jahnstraße 15a, 17489 Greifswald, Germany
contact email	dbecher@uni-greifswald.de
lab head
Andreas Otto
contact affiliation	Institute for Microbiology
contact email	andreas.otto@uni-greifswald.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/2017/10/PXD005262

PRIDE project URI

• PRIDE
  1. PXD005262
     1. Label: PRIDE project
     2. Name: A protocol for global quantification of phosphoproteins combining metabolic labeling and gel-based proteomics