Dataset Information

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

ABSTRACT: 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.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Escherichia Coli

TISSUE(S): Cell Culture

SUBMITTER: Miao-Hsia Lin

LAB HEAD: Miao-Hsia Lin

PROVIDER: PXD001264 | Pride | 2015-09-22

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	04_Ecoli_HAMMOC_1.wiff	Wiff
	04_Ecoli_HAMMOC_1.wiff.scan	Wiff
	05_Ecoli_HAMMOC_2.wiff	Wiff
	05_Ecoli_HAMMOC_2.wiff.scan	Wiff
	130312miao_004_PTS_MC_HAMMOC_1.wiff	Wiff

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Publications

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

Lin Miao-Hsia MH Sugiyama Naoyuki N Ishihama Yasushi Y

Science signaling 20150915 394

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 ...[more]

PMID: 26373674

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Project description:Protein phosphorylation regulated by plant hormone is involved in the coordination of fundamental plant development. Brassinosteroids (BRs), a group of phytohormones, regulated phosphorylation dynamics remains to be delineated in plants. In this study, we performed a mass spectrometry (MS)-based phosphoproteomics to conduct a global and dynamic phosphoproteome profiling across five time points of BR treatment in the period between 5 minutes and 12 hours. MS coupling with phosphopeptide enrichment techniques has become the powerful tool for profiling protein phosphorylation. However, MS-based methods tend to have data consistency and coverage issues. To address these issues, bioinformatics approaches were used to complement the non-detected proteins and recover the dynamics of phosphorylation events. A total of 1,104 unique phosphorylated peptides from 739 unique phosphoproteins were identified. The time-dependent gene ontology (GO) analysis shows the transition of biological processes from signaling transduction to morphogenesis and stress response. The protein-protein interaction analysis found most of identified phosphoproteins have strongly connections with known BR signaling components. The analysis by using Motif-X was performed to identify 15 enriched motifs, 11 of which correspond to 6 known kinase families. To uncover the dynamic activities of kinases, the enriched motifs were combined with phosphorylation profiles and revealed that the substrates of casein kinase 2 and mitogen-activated protein kinase were significantly phosphorylated and dephosphorylated at initial time of BR treatment, respectively. The time-dependent kinase-substrate interaction networks were constructed and showed many substrates are the downstream of other signalings, such as auxin and ABA signalings. Comparing BR responsive phosphoproteome and gene expression data, we found most of phosphorylation changes were not led by gene expression changes. Our results suggested BR signaling not only induces gene expression, but also change protein activation by phosphorylation via various kinases. Through a large-scale dynamic profile of phosphoproteome coupling bioinformatics, a complicated kinase-centered network related to BR-regulated growth was deciphered. The phosphoproteins and phosphosites identified from our study provide a useful dataset for revealing signaling networks of BR regulation, and also expanded our knowledge of protein phosphorylation modification in plants as well as further deal to solve the plant growth problems.