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JPST000100-1 B. subtilis phosphoproteomics [Reanalysis: JPST000100]

ABSTRACT: Protein phosphorylation is a crucial posttranslational modification for regulating cellular processes in bacteria; however, it has not been extensively studied because of technical difficulties in the enrichment of phosphopeptides. We devised an enrichment protocol that enabled the identification of >1000 phosphopeptides from a single bacterial sample. We discovered three high-confidence serine and threonine phosphorylation motifs, as well as 29 other motifs at various levels of confidence, from three distinct bacterial phosphoproteomes. We found that the proline-directed and basophilic phosphorylation motifs that are commonly enriched in eukaryotes were not observed in bacteria. Unlike eukaryotes, bacteria had a low occurrence of both phosphorylation and acetylation in N-terminal phosphopeptides. Because infection of host cells by bacterial pathogens is often accompanied by kinase-mediated phosphorylation events, the differences in phosphorylation preferences between bacteria and eukaryotes revealed by this study could be useful in identifying bacterial-specific targets for future therapies. [Original project description]

ORGANISM(S): Bacillus Subtilis

SUBMITTER: Yasushi Ishihama

PROVIDER: RPXD034742 | JPOST Repository | Sat Apr 01 00:00:00 BST 2023

REPOSITORIES: jPOST

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			Action	DRS
	130926miao_03_Bacillus_MCF_HAM_1.pilotFin.txt	Txt
	130926miao_03_Bacillus_MCF_HAM_1.txt	Txt
	130926miao_04_Bacillus_MCF_HAM_2.pilotFin.txt	Txt
	130926miao_04_Bacillus_MCF_HAM_2.txt	Txt
	130926miao_11_Bacillus_FASP_HAM_1.pilotFin.txt	Txt

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JPST000101-2 K. pneumoniae phosphoproteomics [Reanalysis: JPST000101]

Project description:Protein phosphorylation is a crucial posttranslational modification for regulating cellular processes in bacteria; however, it has not been extensively studied because of technical difficulties in the enrichment of phosphopeptides. We devised an enrichment protocol that enabled the identification of >1000 phosphopeptides from a single bacterial sample. We discovered three high-confidence serine and threonine phosphorylation motifs, as well as 29 other motifs at various levels of confidence, from three distinct bacterial phosphoproteomes. We found that the proline-directed and basophilic phosphorylation motifs that are commonly enriched in eukaryotes were not observed in bacteria. Unlike eukaryotes, bacteria had a low occurrence of both phosphorylation and acetylation in N-terminal phosphopeptides. Because infection of host cells by bacterial pathogens is often accompanied by kinase-mediated phosphorylation events, the differences in phosphorylation preferences between bacteria and eukaryotes revealed by this study could be useful in identifying bacterial-specific targets for future therapies. [Original project description]

2023-03-25 | RPXD034732 | JPOST Repository

JPST000100-2 B. subtilis phosphoproteomics [Reanalysis: JPST000100]

2023-03-25 | RPXD034731 | JPOST Repository

JPST000101-1 K. pneumoniae phosphoproteomics [Reanalysis: JPST000101]

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JPST000099-1 E. coli phosphoproteomics [Reanalysis: JPST000099]

2023-04-01 | RPXD034741 | JPOST Repository

Bacterial phosphoproteome - Systematic profiling of the bacterial phosphoproteome reveals bacterium-specific features of phosphorylation

Project description:Protein phosphorylation is a crucial post-translational modification in bacteria, but has not been extensively studied because of the technical difficulty of phosphopeptide enrichment. We present a new enrichment protocol, approximately 10 times more efficient than conventional approaches in E. coli. This protocol also performed well in B. subtilis and K. pneumoniae, in terms of high coverage and phosphopeptide identification numbers. Moreover, three high-confidence Ser/Thr phosphorylation motifs as well as 29 other motifs at various confidence levels were discovered for the first time, implying that both the position of phospho-acceptor residues and the surrounding sequences are critical for kinase-substrate specificity. As for N-terminal phosphorylation, a low rate of co-occurrence of N-terminal acetylation and acidic residues at the antepenultimate position appears to be prokaryote-specific. The comprehensive prokaryotic phosphoproteomes generated by our new protocol suggest the existence of distinct phosphorylation preferences between prokaryotes and eukaryotes.

2015-09-22 | PXD001264 | Pride

Automated Phosphopeptide Enrichment for Gram Positive Bacteria

Project description:Protein phosphorylation in prokaryotes has gained more attention in recent years as several studies linked it to regulatory and signalling functions indicating an importance similar to protein phosphorylation in eukaryotes. Studies on bacterial phosphorylation have so far been conducted using manual or HPLC-supported phosphopeptide enrichment while automation of phosphopeptide enrichment has been established in eukaryotes, allowing for high throughput sampling. To facilitate the prospect of studying bacterial phosphorylation on a systems‐level we here establish an automated Ser/Thr/Tyr phosphopeptide enrichment workflow on the Agilent AssayMap platform. We present optimized buffer conditions for TiO2 and Fe(III)NTA-IMAC based enrichment, and the most advantageous, species specific starting amount for S. pyogenes, L. monocytogenes, and B. subtilis. Our data represents, to the best of our knowledge, the largest phosphoproteome identified by a single study for each of these bacteria. For higher sample amounts (> 250µg) we observed a superior performance of Fe(III)NTA cartridges, while more peptides were identified from smaller sample amounts using TiO2-based enrichment. Both cartridges largely enrich the same set of phosphopeptides suggesting no improvement of peptide yield from complementary use of both cartridges. Distribution of S/T/Y phosphorylation varied between bacterial strains with threonine being the main site of phosphorylation in S. pyogenes, while in B.subtilis and L. monocytogenes showed the highest percentage of phosphorylation at serine.

2021-08-26 | PXD025459 | Pride

B. subtilis phosphoproteome - Systematic profiling of the bacterial phosphoproteome reveals bacterium-specific features of phosphorylation

Project description:Highly efficient phosphopeptide enrichment developed for bacterial phosphoproteomics. The most comprehensive phosphoproteme maps in both gram-positive/negative bacteria. Discovery of phosphorylation motifs across distinct bacterial species

2015-09-22 | PXD001298 | Pride

K. pneumoniae phosphoproteome - Systematic profiling of the bacterial phosphoproteome reveals bacterium-specific features of phosphorylation

2015-09-22 | PXD001263 | Pride

Parallel Reaction Monitoring on a Q Exactive Mass Spectrometer Increases Reproducibility of Phosphopeptide Detection in Bacterial Phosphoproteomics Measurements

Project description:Increasing number of studies report the relevance of protein Ser/Thr/Tyr phosphorylation in bacterial physiology, yet the analysis of this type of modification in bacteria still presents a considerable challenge. Unlike in eukaryotes, where tens of thousands of phosphorylation events likely occupy more than two thirds of the proteome, the abundance of protein phosphorylation is much lower in bacteria. Even the state-of-the-art phosphopeptide enrichment protocols fail to remove the high background of abundant unmodified peptides, leading to low signal intensity and undersampling of phosphopeptide precursor ions in consecutive data-dependent MS runs. Consequently, large-scale bacterial phosphoproteomic datasets often suffer from poor reproducibility and a high number of missing values. Here we explore the application of parallel reaction monitoring (PRM) on a Q Exactive mass spectrometer in bacterial phosphoproteome analysis, focusing especially on run-to-run sampling reproducibility. In multiple measurements of identical phosphopeptide-enriched samples, we show that PRM outperforms DDA in terms of detection frequency, reaching almost complete sampling efficiency, compared to 20% in DDA. We observe a similar trend over multiple rounds of (heterogeneous) phosphopeptide-enriched samples and conclude that PRM is a method of choice in bacterial phosphoproteomics projects where reproducible detection and quantification of a relatively small set of phosphopeptides is desired.

2018-06-05 | PXD008211 | Pride

A compendium of RNA-binding motifs for analysis of gene regulation

Project description:RNA binding proteins are key regulators of gene expression, yet only a small fraction of these proteins has been functionally characterized. Here, we report the first large-scale analysis of the RNA motifs recognised by RNA binding proteins, encompassing 205 distinct proteins from 24 diverse eukaryotes. The sequence specificities of RBPs display deep evolutionary conservation, such that the recognition preferences for a large fraction of metazoan RNA binding proteins can be inferred from the sequences of their binding domains. The motifs we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA binding proteins in both normal physiology and in human disease. These data provide an unprecedented global view of RBPs and their targets and constitute an invaluable resource for defining post-transcriptional regulatory mechanisms in eukaryotes. Here, we analyze the RNA-binding preferences of 205 distinct RNA-binding proteins from 24 different eukaryotes, using RNAcompete. In this assay, purified GST-tagged RBPs are incubated with an excess of RNA pool and bound RNA from individual pulldowns are directly labeled, hybridized to a custom Agilent 244K microarray, and analyzed computationally to identify RNA-binding motifs.

2013-05-10 | E-GEOD-41235 | biostudies-arrayexpress

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