Proteomics

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A protocol for global quantification of phosphoproteins combining metabolic labeling and gel-based proteomics


ABSTRACT: 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.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Bacillus Pumilus

SUBMITTER: Andreas Otto  

LAB HEAD: Dörte Becher

PROVIDER: PXD005262 | Pride | 2017-10-02

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
AB_allebeide_interact.ipro.pep.xml Pepxml
AC_beideinteract.ipro.pep.xml Pepxml
AE_beide_interact.ipro.pep.xml Pepxml
AH_beide_interact.ipro.pep.xml Pepxml
AK_beide_interact.ipro.pep.xml Pepxml
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Publications

Global quantification of phosphoproteins combining metabolic labeling and gel-based proteomics in B. pumilus.

Hentschker Christian C   Dewald Carolin C   Otto Andreas A   Büttner Knut K   Hecker Michael M   Becher Dörte D  

Electrophoresis 20171017 2


Differential proteomics targeting the protein abundance is commonly used to follow changes in biological systems. Differences in localization and degree of post-translational modifications of proteins including phosphorylations are of tremendous interest due to the anticipated role in molecular regulatory processes. Because of their particular low abundance in prokaryotes, identification and quantification of protein phosphorylation is traditionally performed by either comparison of spot intensi  ...[more]

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