Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.12 (PKG)
 2,515 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Selective detection of phosphopeptides from proteolytic digests is a challenging and highly relevant task in many proteomics applications. Often phosphopeptides are present in small amounts and need selective isolation or enrichment before identification. Here we report a novel automated method for the enrichment of phosphopeptides from complex mixtures. The method employs a two-dimensional column setup, with titanium oxide-based solid-phase material (Titansphere) as the first dimension and reversed-phase material as the second dimension. Phosphopeptides are separated from nonphosphorylated peptides by trapping them under acidic conditions on a TiO(2) precolumn. Nonphosphorylated peptides break through and are trapped on a reversed-phase precolumn after which they are analyzed by nanoflow LC-ESI-MS/MS. Subsequently, phosphopeptides are desorbed from the TiO(2) column under alkaline conditions, reconcentrated onto the reversed-phase precolumn, and analyzed by nanoflow LC-ESI-MS/MS. The selectivity and practicality of using TiO(2) precolumns for trapping phosphopeptides are demonstrated via the analysis of a model peptide RKISASEF, in a 1:1 mixture of a non- and a monophosphorylated form. A sample of 125 fmol of the phosphorylated peptide could easily be isolated from the nonphosphorylated peptide with a recovery above 90%. In addition, proteolytic digests of three different autophosphorylation forms of the 153-kDa homodimeric cGMP-dependent protein kinase are analyzed. From proteolytic digests of the fully autophosphorylated protein at least eight phosphorylation sites are identified, including two previously uncharacterized sites, namely, Ser-26 and Ser-44. Ser-26 is characterized as a minor phosphorylation site in purified PKG samples, while Ser-44 is identified as a novel in vitro autophosphorylation target. These results clearly show that TiO(2) has strong affinity for phosphorylated peptides, and thus, we conclude that this material has a high potential in the field of phosphoproteomics.
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PMID:Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. 1525 27

To gain insights into differences in the virulence among T. gondii strains at the post-translational level, we conducted a quantitative analysis of the phosphoproteome profile of T. gondii strains belonging to three different genotypes. Phosphopeptides from three strains, type I (RH strain), type II (PRU strain) and ToxoDB#9 (PYS strain), were enriched by titanium dioxide (TiO2) affinity chromatography and quantified using iTRAQ technology. A total of 1,441 phosphopeptides, 1,250 phosphorylation sites and 759 phosphoproteins were detected. In addition, 392, 298, and 436 differentially expressed phosphoproteins (DEPs) were identified in RH strain when comparing RH/PRU strains, in PRU strain when comparing PRU/PYS strains, and in PYS strain when comparing PYS/RH strains, respectively. Functional characterization of the DEPs using GO, KEGG, and STRING analyses revealed marked differences between the three strains. In silico kinase substrate motif analysis of the DEPs revealed three (RxxS, SxxE, and SxxxE), three (RxxS, SxxE, and SP), and five (SxxE, SP, SxE, LxRxxS, and RxxS) motifs in RH strain when comparing RH/PRU strains, in PRU strain when comparing PRU/PYS, and in PYS strain when comparing PYS/RH strains, respectively. This suggests that multiple overrepresented protein kinases including PKA, PKG, CKII, IKK, and MAPK could be involved in such a difference between T. gondii strains. Kinase associated network analysis showed that ROP5, ROP16, and cell-cycle-associated protein kinase CDK were the most connected kinase peptides. Our data reveal significant changes in the abundance of phosphoproteins between T. gondii genotypes, which explain some of the mechanisms that contribute to the virulence heterogeneity of this parasite.
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PMID:iTRAQ-Based Global Phosphoproteomics Reveals Novel Molecular Differences Between Toxoplasma gondii Strains of Different Genotypes. 3150 80