Dataset Information

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

ABSTRACT: 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

INSTRUMENT(S): TripleTOF 5600, Q Exactive

ORGANISM(S): Klebsiella Pneumoniae Subsp. Pneumoniae Ntuh-k2044

TISSUE(S): Cell Culture

SUBMITTER: Miao-Hsia Lin

LAB HEAD: Miao-Hsia Lin

PROVIDER: PXD001263 | Pride | 2015-09-22

REPOSITORIES: Pride

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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.

Project description:Albeit much less abundant than Ser/Thr phosphorylation (pSer/pThr), Tyr phosphorylation (pTyr) is considered a hallmark in cellular signal transduction. However, its analysis remains a challenge. The conventional immunopurification (IP) approach using antibodies pan-specific to pTyr sites is known to have low sensitivity, poor reproducibility and high cost. SH2 domain-derived pTyr-superbinder is a good replacement of pTyr antibody for the specific enrichment of pTyr peptides for phosphoproteomics analysis. In this study, we aim to optimize the approach using SH2 superbinder for tyrosine phosphoproteome analysis. The present work covalently immobilized SH2 superbinders to agrose beads, designed and evaluated four different SH2 superbinder based experimental workflows for phosphotyrosine analysis. After made a head to head comparison of them, we observed that the combined strategy employing Ti4+-IMAC and covalently immobilized SH2 superbinder enrichment in sequence generated the largest pTyr peptide dataset (3519) with the best selectivity (90%). Next the optimal method was applied for pTyr profiling from normal mouse tissues, and resulted in the identification of 197 pTyr sites (of which 73 were novel) from 5 mg protein digests, which validated its high sensitivity. An comparison with antibody 4G10 by isolating pTyr peptides from 5 mg unstimulated Jurkat cell digests were made, our optimal strategy identified 343 while the antibody 4G10 based method identified 242 pTyr sites, respectively, further confirmed the robustness of the method. Finally, the optimal strategy was applied for quantitative pTyr phosphorylation analysis of EGF stimulated/unstimulated HeLa cells, 261 pTyr sites were successfully quantified from 5 mg stable-isotope dimethyl labling protein digests. In general, the combination of Ti4+-IMAC and SH2 superbinder enrichment in sequence generated a facial and robust strategy for qualitative and quantitative analysis of pTyr proteome.

Dataset Information

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

Publications

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

Similar Datasets

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